Cyclopropanecarboxamide modulators of cystic fibrosis transmembrane conductance regulator

ABSTRACT

The present invention relates to new cyclopropanecarboxamide modulators of cystic fibrosis transmembrane conductance regulator proteins, pharmaceutical compositions thereof, and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application filed under 35 U.S.C.371 of International Patent Application No. PCT/US2015/067544, filedDec. 27, 2015, which claims the benefit of priority of U.S. ProvisionalPatent Application No. 62/098,735, filed Dec. 31, 2014, the disclosureof which is hereby incorporated by reference as if written herein in itsentirety.

Disclosed herein are new cyclopropanecarboxamide compounds andcompositions and their application as pharmaceuticals for the treatmentof disorders. Methods of modulation of cystic fibrosis transmembraneconductance regulator activity in a subject are also provided for thetreatment of disorders such as cystic fibrosis, sarcoglycanopathies,Brody's disease, cathecolaminergic polymorphic ventricular tachycardia,limb girdle muscular dystrophy, asthma, smoke induced chronicobstructive pulmonary disorder, chronic bronchitis, rhinosinusitis,constipation, pancreatitis, pancreatic insufficiency, male infertilitycaused by congenital bilateral absence of the vas deferens (CBAVD), mildpulmonary disease, idiopathic pancreatitis, allergic bronchopulmonaryaspergillosis (ABPA), liver disease, hereditary emphysema, hereditaryhemochromatosis, coagulation-fibrinolysis deficiencies, such as proteinC deficiency, type 1 hereditary angioedema, lipid processingdeficiencies, such as familial hypercholesterolemia, type 1chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, suchas I-cell disease/pseudo-Hurler, mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,polyendocrinopathy/hyperinsulinemia, diabetes mellitus, Laron dwarfism,myeloperoxidase deficiency, primary hypoparathyroidism, melanoma,glycanosis CDG type 1, congenital hyperthyroidism, osteogenesisimperfecta, hereditary hypofibrinogenemia, ACT deficiency, diabetesinsipidus (DI), neurohypophyseal DI, nephrogenic DI, Charcot-Marie toothsyndrome, Pelizaeus-Merzbacher disease, neurodegenerative diseases suchas Alzheimer's disease, Parkinson's disease, amyotrophic lateralsclerosis, progressive supranuclear palsy, Pick's disease, polyglutamineneurological disorders such as Huntington's, spinocerebellar ataxia typeI, spinal and bulbar muscular atrophy, dentatombral pallidoluysian, andmyotonic dystrophy, as well as spongifiorm encephalopathies, such ashereditary Creutzfeldt-Jakob disease (due to prion protein processingdefect), Fabry disease, Gerstrnarm-Straussler-Scheinker syndrome,chronic obstructive pulmonary disorder, dry-eye disease, or Sjogren'sdisease, osteoporosis, osteopenia, bone healing and bone growth(including bone repair, bone regeneration, reducing bone resorption andincreasing bone deposition), Gorham's Syndrome, chloride channelopathiessuch as myotonia congenita (Thomson and Becker forms), Bartter'ssyndrome type III, Dent's disease, hyperekplexia, epilepsy, lysosomalstorage disease, Angelman syndrome, and primary ciliary dyskinesia(PCD), a term for inherited disorders of the structure and/or functionof cilia, including PCD with situs inversus (also known as Kartagenersyndrome), PCD without situs inversus, and ciliary aplasia.

VX-661 (CAS #: 1152311-62-0;1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(2-hydroxy-1,1-dimethylethyl)-1H-indol-5-yl]-cyclopropanecarboxamide).VX-661 is a cystic fibrosis transmembrane conductance regulatormodulator. VX-661 is currently under investigation for the treatment ofcystic fibrosis. VX-661 has also shown promise in treatingsarcoglycanopathies, Brody's disease, cathecolaminergic polymorphicventricular tachycardia, limb girdle muscular dystrophy, asthma, smokeinduced chronic obstructive pulmonary disorder, chronic bronchitis,rhinosinusitis, constipation, pancreatitis, pancreatic insufficiency,male infertility caused by congenital bilateral absence of the vasdeferens (CBAVD), mild pulmonary disease, idiopathic pancreatitis,allergic bronchopulmonary aspergillosis (ABPA), liver disease,hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulinemia, diabetesmellitus, Laron dwarfism, myeloperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, congenitalhyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia,ACT deficiency, diabetes insipidus (DI), neurohypophyseal DI,nephrogenic DI, Charcot-Marie tooth syndrome, Pelizaeus-Merzbacherdisease, neurodegenerative diseases such as Alzheimer's disease,Parkinson's disease, amyotrophic lateral sclerosis, progressivesupranuclear palsy, Pick's disease, polyglutamine neurological disorderssuch as Huntington's, spinocerebellar ataxia type I, spinal and bulbarmuscular atrophy, dentatombral pallidoluysian, and myotonic dystrophy,as well as spongifiorm encephalopathies, such as hereditaryCreutzfeldt-Jakob disease (due to prion protein processing defect),Fabry disease, Gerstrnarm-Straussler-Scheinker syndrome, chronicobstructive pulmonary disorder, dry-eye disease, or Sjogren's disease,osteoporosis, osteopenia, bone healing and bone growth (including bonerepair, bone regeneration, reducing bone resorption and increasing bonedeposition), Gorham's Syndrome, chloride channelopathies such asmyotonia congenita (Thomson and Becker forms), Bartter's syndrome typeIII, Dent's disease, hyperekplexia, epilepsy, lysosomal storage disease,Angelman syndrome, and primary ciliary dyskinesia (PCD), a term forinherited disorders of the structure and/or function of cilia, includingPCD with situs inversus (also known as Kartagener syndrome), PCD withoutsitus inversus, and ciliary aplasia. WO 2014086687; WO2013185112.

VX-661 is likely subject to extensive CYP450-mediated oxidativemetabolism. These, as well as other metabolic transformations, occur inpart through polymorphically-expressed enzymes, exacerbatinginterpatient variability. Additionally, some metabolites of VX-661 mayhave undesirable side effects. In order to overcome its short half-life,the drug likely must be taken several times per day, which increases theprobability of patient incompliance and discontinuance.

Deuterium Kinetic Isotope Effect

In order to eliminate foreign substances such as therapeutic agents, theanimal body expresses various enzymes, such as the cytochrome P450enzymes (CYPs), esterases, proteases, reductases, dehydrogenases, andmonoamine oxidases, to react with and convert these foreign substancesto more polar intermediates or metabolites for renal excretion. Suchmetabolic reactions frequently involve the oxidation of acarbon-hydrogen (C—H) bond to either a carbon-oxygen (C—O) or acarbon-carbon (C—C) π-bond. The resultant metabolites may be stable orunstable under physiological conditions, and can have substantiallydifferent pharmacokinetic, pharmacodynamic, and acute and long-termtoxicity profiles relative to the parent compounds. For most drugs, suchoxidations are generally rapid and ultimately lead to administration ofmultiple or high daily doses.

The relationship between the activation energy and the rate of reactionmay be quantified by the Arrhenius equation, k=Ae^(−Eact/RT). TheArrhenius equation states that, at a given temperature, the rate of achemical reaction depends exponentially on the activation energy(E_(act)).

The transition state in a reaction is a short lived state along thereaction pathway during which the original bonds have stretched to theirlimit. By definition, the activation energy E_(act) for a reaction isthe energy required to reach the transition state of that reaction. Oncethe transition state is reached, the molecules can either revert to theoriginal reactants, or form new bonds giving rise to reaction products.A catalyst facilitates a reaction process by lowering the activationenergy leading to a transition state. Enzymes are examples of biologicalcatalysts.

Carbon-hydrogen bond strength is directly proportional to the absolutevalue of the ground-state vibrational energy of the bond. Thisvibrational energy depends on the mass of the atoms that form the bond,and increases as the mass of one or both of the atoms making the bondincreases. Since deuterium (D) has twice the mass of protium (¹H), a C-Dbond is stronger than the corresponding C-¹H bond. If a C-¹H bond isbroken during a rate-determining step in a chemical reaction (i.e. thestep with the highest transition state energy), then substituting adeuterium for that protium will cause a decrease in the reaction rate.This phenomenon is known as the Deuterium Kinetic Isotope Effect (DKIE).The magnitude of the DKIE can be expressed as the ratio between therates of a given reaction in which a C-¹H bond is broken, and the samereaction where deuterium is substituted for protium. The DKIE can rangefrom about 1 (no isotope effect) to very large numbers, such as 50 ormore. Substitution of tritium for hydrogen results in yet a strongerbond than deuterium and gives numerically larger isotope effects

Deuterium (²H or D) is a stable and non-radioactive isotope of hydrogenwhich has approximately twice the mass of protium (¹H), the most commonisotope of hydrogen. Deuterium oxide (D₂O or “heavy water”) looks andtastes like H₂O, but has different physical properties.

When pure D₂O is given to rodents, it is readily absorbed. The quantityof deuterium required to induce toxicity is extremely high. When about0-15% of the body water has been replaced by D₂O, animals are healthybut are unable to gain weight as fast as the control (untreated) group.When about 15-20% of the body water has been replaced with D₂O, theanimals become excitable. When about 20-25% of the body water has beenreplaced with D₂O, the animals become so excitable that they go intofrequent convulsions when stimulated. Skin lesions, ulcers on the pawsand muzzles, and necrosis of the tails appear. The animals also becomevery aggressive. When about 30% of the body water has been replaced withD₂O, the animals refuse to eat and become comatose. Their body weightdrops sharply and their metabolic rates drop far below normal, withdeath occurring at about 30 to about 35% replacement with D₂O. Theeffects are reversible unless more than thirty percent of the previousbody weight has been lost due to D₂O. Studies have also shown that theuse of D₂O can delay the growth of cancer cells and enhance thecytotoxicity of certain antineoplastic agents.

Deuteration of pharmaceuticals to improve pharmacokinetics (PK),pharmacodynamics (PD), and toxicity profiles has been demonstratedpreviously with some classes of drugs. For example, the DKIE was used todecrease the hepatotoxicity of halothane, presumably by limiting theproduction of reactive species such as trifluoroacetyl chloride.However, this method may not be applicable to all drug classes. Forexample, deuterium incorporation can lead to metabolic switching.Metabolic switching occurs when xenogens, sequestered by Phase Ienzymes, bind transiently and re-bind in a variety of conformationsprior to the chemical reaction (e.g., oxidation). Metabolic switching isenabled by the relatively vast size of binding pockets in many Phase Ienzymes and the promiscuous nature of many metabolic reactions.Metabolic switching can lead to different proportions of knownmetabolites as well as altogether new metabolites. This new metabolicprofile may impart more or less toxicity. Such pitfalls are non-obviousand are not predictable a priori for any drug class.

VX-661 is a cystic fibrosis transmembrane conductance regulatormodulator. The carbon-hydrogen bonds of VX-661 contain a naturallyoccurring distribution of hydrogen isotopes, namely ¹H or protium (about99.9844%), ²H or deuterium (about 0.0156%), and ³H or tritium (in therange between about 0.5 and 67 tritium atoms per 10¹⁸ protium atoms).Increased levels of deuterium incorporation may produce a detectableDeuterium Kinetic Isotope Effect (DKIE) that could affect thepharmacokinetic, pharmacologic and/or toxicologic profiles of suchVX-661 in comparison with the compound having naturally occurring levelsof deuterium.

Based on discoveries made in our laboratory, as well as considering theliterature, VX-661 is likely metabolized in humans at the cyclopropylring, the geminal methyl groups, the hydroxyl methylene group, and the2-hydroxy butyl group. The current approach has the potential to preventmetabolism at these sites. Other sites on the molecule may also undergotransformations leading to metabolites with as-yet-unknownpharmacology/toxicology. Limiting the production of these metaboliteshas the potential to decrease the danger of the administration of suchdrugs and may even allow increased dosage and/or increased efficacy. Allof these transformations can occur through polymorphically-expressedenzymes, exacerbating interpatient variability. Further, some disordersare best treated when the subject is medicated around the clock or foran extended period of time. For all of the foregoing reasons, a medicinewith a longer half-life may result in greater efficacy and cost savings.Various deuteration patterns can be used to (a) reduce or eliminateunwanted metabolites, (b) increase the half-life of the parent drug, (c)decrease the number of doses needed to achieve a desired effect, (d)decrease the amount of a dose needed to achieve a desired effect, (e)increase the formation of active metabolites, if any are formed, (0decrease the production of deleterious metabolites in specific tissues,and/or (g) create a more effective drug and/or a safer drug forpolypharmacy, whether the polypharmacy be intentional or not. Thedeuteration approach has the strong potential to slow the metabolism ofVX-661 and attenuate interpatient variability.

Novel compounds and pharmaceutical compositions, certain of which havebeen found to modulate cystic fibrosis transmembrane conductanceregulator have been discovered, together with methods of synthesizingand using the compounds, including methods for the treatment of cysticfibrosis transmembrane conductance regulator-mediated disorders in apatient by administering the compounds.

In certain embodiments of the present invention, compounds havestructural Formula I:

or a salt thereof, wherein:

R₁-R₃ and R₆-R₂₃ are independently selected from the group consisting ofhydrogen and deuterium;

R₄-R₅ are independently selected from the group consisting of —CH₃,—CH₂D, —CD₂H, and —CD₃; and

at least one of R₁-R₂₃ is deuterium or contains deuterium.

Also provided are enantiomers of compounds of Formula I, designatedFormulas Ia and Ib:

In certain embodiments of the present invention, compounds havestructural Formula Ia:

or a salt thereof, wherein:

R₁-R₃ and R₆-R₂₃ are independently selected from the group consisting ofhydrogen and deuterium;

R₄-R₅ are independently selected from the group consisting of —CH₃,—CH₂D, —CD₂H, and —CD₃; and

at least one of R₁-R₂₃ is deuterium or contains deuterium.

In certain embodiments, R₁, R₆, R₉, and R₁₆ are hydrogen.

In certain embodiments, R₂ and R₃ are deuterium.

In certain embodiments, R₄ and R₅ are —CD₃.

In certain embodiments, R₂ and R₃ are deuterium; and R₄ and R₅ are —CD₃.

In certain embodiments, R₇ and R₈ are deuterium.

In certain embodiments, R₇, R₈, and R₁₀ are deuterium.

In certain embodiments, R₂, R₃, R₇, and R₈ are deuterium.

In certain embodiments, R₂, R₃, R₇, R₈, and R₁₀ are deuterium.

In certain embodiments, R₇ and R₈ are deuterium; and R₄ and R₅ are —CD₃.

In certain embodiments, R₇, R₈, and R₁₀ are deuterium; and R₄ and R₅ are—CD₃.

In certain embodiments, R₂, R₃, R₇, and R₈ are deuterium; and R₄ and R₅are —CD₃.

In certain embodiments, R₂, R₃, R₇, R₈, and R₁₀ are deuterium; and R₄and R₅ are —CD₃.

In certain embodiments, R₁₇-R₂₀ are deuterium.

In certain embodiments, R₁₇-R₂₀ are deuterium; and R₄ and R₅ are —CD₃.

In certain embodiments, R₂, R₃, and R₁₇-R₂₀ are deuterium.

In certain embodiments, R₂, R₃, and R₁₇-R₂₀ are deuterium; and R₄ and R₅are —CD₃.

In certain embodiments, R₇, R₈, and R₁₇-R₂₀ are deuterium.

In certain embodiments, R₇, R₈, R₁₀, and R₁₇-R₂₀ are deuterium.

In certain embodiments, R₂, R₃, R₇, R₈, and R₁₇-R₂₀ are deuterium.

In certain embodiments, R₂, R₃, R₇, R₈, R₁₀, and R₁₇-R₂₀ are deuterium.

In certain embodiments, R₇, R₈, and R₁₇-R₂₀ are deuterium; and R₄ and R₅are —CD₃.

In certain embodiments, R₇, R₈, R₁₀, and R₁₇-R₂₀ are deuterium; and R₄and R₅ are —CD₃.

In certain embodiments, R₂, R₃, R₇, R₈, and R₇₁-R₂₀ are deuterium; andR₄ and R₅ are —CD₃.

In certain embodiments, R₂, R₃, R₇, R₈, R₁₀, and R₁₇-R₂₀ are deuterium;and R₄ and R₅ are —CD₃.

In certain embodiments, R₂₁-R₂₃ and R₁₃-R₁₅ are hydrogen.

Also provided herein are embodiments according to each of theembodiments above, wherein:

every other substituent among R₁-R₃ and R₆-R₂₃ not specified asdeuterium is hydrogen; and

if R₄ and R₅ are not specified to be —CD₃, then they are —CH₃.

In certain embodiments are provided compounds as disclosed herein,wherein at least one of R₁-R₂₃ independently has deuterium enrichment ofno less than about 1%. In certain embodiments are provided compounds asdisclosed herein, wherein at least one of R₁-R₂₃ independently hasdeuterium enrichment of no less than about 10%. In certain embodimentsare provided compounds as disclosed herein, wherein at least one ofR₁-R₂₃ independently has deuterium enrichment of no less than about 50%.In certain embodiments are provided compounds as disclosed herein,wherein at least one of R₁-R₂₃ independently has deuterium enrichment ofno less than about 90%. In certain embodiments are provided compounds asdisclosed herein, wherein at least one of R₁-R₂₃ independently hasdeuterium enrichment of no less than about 95%. In certain embodimentsare provided compounds as disclosed herein, wherein at least one ofR₁-R₂₃ independently has deuterium enrichment of no less than about 98%.

Also provided is a compound chosen from the Examples and compoundsdisclosed herein.

The compounds as disclosed herein may also contain less prevalentisotopes for other elements, including, but not limited to, ¹³C or ¹⁴Cfor carbon, ³³S, ³⁴S, or ³⁶S for sulfur, ¹⁵N for nitrogen, and ¹⁷O or¹⁸O for oxygen.

In certain embodiments, the compound disclosed herein may expose apatient to a maximum of about 0.000005% D₂O or about 0.00001% DHO,assuming that all of the C-D bonds in the compound as disclosed hereinare metabolized and released as D₂O or DHO. In certain embodiments, thelevels of D₂O shown to cause toxicity in animals is much greater thaneven the maximum limit of exposure caused by administration of thedeuterium enriched compound as disclosed herein. Thus, in certainembodiments, the deuterium-enriched compound disclosed herein should notcause any additional toxicity due to the formation of D₂O or DHO upondrug metabolism.

In certain embodiments are provided compounds as disclosed hereinwherein each position represented as D has deuterium enrichment of noless than about 1%. In certain embodiments are provided compounds asdisclosed herein wherein each position represented as D has deuteriumenrichment of no less than about 10%. In certain embodiments areprovided compounds as disclosed herein wherein each position representedas D has deuterium enrichment of no less than about 50%. In certainembodiments are provided compounds as disclosed herein wherein eachposition represented as D has deuterium enrichment of no less than about90%. In certain embodiments are provided compounds as disclosed hereinwherein each position represented as D has deuterium enrichment of noless than about 95%. In certain embodiments are provided compounds asdisclosed herein wherein each position represented as D has deuteriumenrichment of no less than about 98%.

In certain embodiments, the deuterated compounds disclosed hereinmaintain the beneficial aspects of the corresponding non-isotopicallyenriched molecules while substantially increasing the maximum tolerateddose, decreasing toxicity, increasing the half-life (T_(1/2)), loweringthe maximum plasma concentration (C_(max)) of the minimum efficaciousdose (MED), lowering the efficacious dose and thus decreasing thenon-mechanism-related toxicity, and/or lowering the probability ofdrug-drug interactions.

Compounds disclosed herein possess useful cystic fibrosis transmembraneconductance regulator modulating activity, and may be used in thetreatment or prophylaxis of a disorder in which cystic fibrosistransmembrane conductance regulator proteins play an active role. Thus,certain embodiments also provide pharmaceutical compositions comprisingone or more compounds disclosed herein together with a pharmaceuticallyacceptable carrier, as well as methods of making and using the compoundsand compositions. Certain embodiments provide methods for modulatingcystic fibrosis transmembrane conductance regulator proteins. Otherembodiments provide methods for treating a cystic fibrosis transmembraneconductance regulator-mediated disorder in a patient in need of suchtreatment, comprising administering to the patient a therapeuticallyeffective amount of a compound or composition according to the presentinvention. Also provided is the use of certain compounds disclosedherein for use in the manufacture of a medicament for the prevention ortreatment of a disorder ameliorated by the modulation of cystic fibrosistransmembrane conductance regulator proteins.

Also provided is a method of treatment of a cystic fibrosistransmembrane conductance regulator-mediated disorder comprising theadministration of a therapeutically effective amount of a compound, or asalt thereof, as disclosed herein to a patient in need thereof.

In certain embodiments, the disorder is selected from the groupconsisting of cystic fibrosis, sarcoglycanopathies, Brody's disease,cathecolaminergic polymorphic ventricular tachycardia, limb girdlemuscular dystrophy, asthma, smoke induced chronic obstructive pulmonarydisorder, chronic bronchitis, rhinosinusitis, constipation,pancreatitis, pancreatic insufficiency, male infertility caused bycongenital bilateral absence of the vas deferens (CBAVD), mild pulmonarydisease, idiopathic pancreatitis, allergic bronchopulmonaryaspergillosis (ABPA), liver disease, hereditary emphysema, hereditaryhemochromatosis, coagulation-fibrinolysis deficiencies, such as proteinC deficiency, type 1 hereditary angioedema, lipid processingdeficiencies, such as familial hypercholesterolemia, type 1chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, suchas I-cell disease/pseudo-Hurler, mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,polyendocrinopathy/hyperinsulinemia, diabetes mellitus, Laron dwarfism,myeloperoxidase deficiency, primary hypoparathyroidism, melanoma,glycanosis CDG type 1, congenital hyperthyroidism, osteogenesisimperfecta, hereditary hypofibrinogenemia, ACT deficiency, diabetesinsipidus (DI), neurohypophyseal DI, nephrogenic DI, Charcot-Marie toothsyndrome, Pelizaeus-Merzbacher disease, neurodegenerative diseases suchas Alzheimer's disease, Parkinson's disease, amyotrophic lateralsclerosis, progressive supranuclear palsy, Pick's disease, polyglutamineneurological disorders such as Huntington's, spinocerebellar ataxia typeI, spinal and bulbar muscular atrophy, dentatombral pallidoluysian, andmyotonic dystrophy, as well as spongifiorm encephalopathies, such ashereditary Creutzfeldt-Jakob disease (due to prion protein processingdefect), Fabry disease, Gerstrnarm-Straussler-Scheinker syndrome,chronic obstructive pulmonary disorder, dry-eye disease, or Sjogren'sdisease, osteoporosis, osteopenia, bone healing and bone growth(including bone repair, bone regeneration, reducing bone resorption andincreasing bone deposition), Gorham's Syndrome, chloride channelopathiessuch as myotonia congenita (Thomson and Becker forms), Bartter'ssyndrome type III, Dent's disease, hyperekplexia, epilepsy, lysosomalstorage disease, Angelman syndrome, and primary ciliary dyskinesia(PCD), a term for inherited disorders of the structure and/or functionof cilia, including PCD with situs inversus (also known as Kartagenersyndrome), PCD without situs inversus, and ciliary aplasia.

In certain embodiments, the disorder is cystic fibrosis.

In certain embodiments, method of treatment of a cystic fibrosistransmembrane conductance regulator-mediated disorder further comprisesthe administration of an additional therapeutic agent.

In certain embodiments, the additional therapeutic agent is selectedfrom the group consisting of antibiotics, bronchodilators,anticholinergics, DNase, mucolytics, nonsteroidal anti-inflammatorydrugs, mast cell stabilizers, corticosteroids, and enzyme replacements.

In certain embodiments, the additional therapeutic agent is anantibiotic selected from the group consisting of amikacin, amoxicillin,ampicillin, arsphenamine, azithromycin, aztreonam, azlocillin,bacitracin, carbenicillin, cefaclor, cefadroxil, cefamandole, cefazolin,cephalexin, cefdinir, cefditorin, cefepime, cefixime, cefoperazone,cefotaxime, cefoxitin, cefpodoxime, cefprozil, ceftazidime, ceftibuten,ceftizoxime, ceftriaxone, cefuroxime, chloramphenicol, cilastin,ciprofloxacin, clarithromycin, clindamycin, cloxacillin, colistin,dalfopristan, demeclocycline, dicloxacillin, dirithromycin, doxycycline,erythromycin, enafloxacin, ertepenem, ethambutol, flucloxacillin,fosfomycin, furazolidone, gatifloxacin, geldanamycin, gentamicin,herbimicin, imipenem, isoniazide, kanamicin, levofloxacin, linezolid,lomefloxacin, loracarbef, mafenide, moxifloxacin, meropenem,metronidazole, mezlocillin, minocycline, mupirozin, nafcillin, neomycin,netilmicin, nitrofurantoin, norfloxacin, ofloxacin, oxytetracycline,penicillin, piperacillin, platensimycin, polymixin B, prontocil,pyrazinamide, quinupristine, retapamulin, rifampin, roxithromycin,spectinomycin, streptomycin, sulfacetamide, sulfamethizole,sulfamethoxazole, teicoplanin, telithromycin, tetracycline, ticarcillin,tobramycin, trimethoprim, troleandomycin, trovafloxacin, and vancomycin.

In certain embodiments, the additional therapeutic agent is abronchodilator selected from the group consisting of salbutamol,levosalbutamol, terbutaline, pirbuterol, procaterol, metaproterenol,fenoterol, bitolterol mesylate, reproterol, salmeterol, formoterol,bambuterol, clenbuterol, and indacaterol.

In certain embodiments, the additional therapeutic agent is ananticholinergic selected from the group consisting of oxyphencyclimine,camylofin, mebeverine, trimebutine, rociverine, dicycloverine,dihexyverine, difemerine, piperidolate, benzilone, glycopyrronium,oxyphenonium, penthienate, propantheline, otilonium bromide,methantheline, tridihexethyl, isopropamide, hexocyclium, poldine,mepenzolate, bevonium, pipenzolate, biphemanil,(2-benzhydryloxyethyl)diethyl-methylammonium iodide, tiemonium iodide,prifinium bromide, timepidium bromide, tiotropium bromide, ipratropiumbromide, and fenpiverinium.

In certain embodiments, the additional therapeutic agent is a DNaseselected from the group consisting of DNase 1 enzyme, pulmozne, anddornase alfa.

In certain embodiments, the additional therapeutic agent is a mucolyticselected from the group consisting of acetylcysteine, ambroxol,carbocisteine, erdosteine, and mecysteine.

In certain embodiments, the additional therapeutic agent is anonsteroidal anti-inflammatory drug selected from the group consistingof lumiracoxib, aceclofenac, acemetacin, amoxiprin, aspirin,azapropazone, benorilate, bromfenac, carprofen, celecoxib, cholinemagnesium salicylate, diclofenac, diflunisal, etodolac, etoracoxib,faislamine, fenbuten, fenoprofen, flurbiprofen, ibuprofen, indometacin,ketoprofen, ketorolac, lornoxicam, loxoprofen, meloxicam, meclofenamicacid, mefenamic acid, meloxicam, metamizole, methyl salicylate,magnesium salicylate, nabumetone, naproxen, nimesulide, oxyphenbutazone,parecoxib, phenylbutazone, piroxicam, salicyl salicylate, sulindac,sulfinprazone, suprofen, tenoxicam, tiaprofenic acid, and tolmetin.

In certain embodiments, the additional therapeutic agent is a mast cellstabilizer selected from the group consisting of cromolyn sodium andnedocromil sodium.

In certain embodiments, the additional therapeutic agent is acorticosteroid selected from the group consisting of prednisone,prednisolne, hydrocortisone, beclometasone, ciclesonide, budesonide,flunisolide, betamethasone, fluticasone, triamcinolone, and mometasone.

In certain embodiments, the additional therapeutic agent is an enzymereplacement selected from the group consisting of pancrelipase, lipase,protease, and amylase.

In certain embodiments, method of treatment of a cystic fibrosistransmembrane conductance regulator-mediated disorder further results inat least one effect selected from the group consisting of:

-   -   a) decreased inter-individual variation in plasma levels of the        compound or a metabolite thereof as compared to the        non-isotopically enriched compound;    -   b) increased average plasma levels of the compound per dosage        unit thereof as compared to the non-isotopically enriched        compound;    -   c) decreased average plasma levels of at least one metabolite of        the compound per dosage unit thereof as compared to the        non-isotopically enriched compound;    -   d) increased average plasma levels of at least one metabolite of        the compound per dosage unit thereof as compared to the        non-isotopically enriched compound; and    -   e) an improved clinical effect during the treatment in the        subject per dosage unit thereof as compared to the        non-isotopically enriched compound.

In certain embodiments, method of treatment of a cystic fibrosistransmembrane conductance regulator-mediated disorder further results inat least two effects selected from the group consisting of:

-   -   a) decreased inter-individual variation in plasma levels of the        compound or a metabolite thereof as compared to the        non-isotopically enriched compound;    -   b) increased average plasma levels of the compound per dosage        unit thereof as compared to the non-isotopically enriched        compound;    -   c) decreased average plasma levels of at least one metabolite of        the compound per dosage unit thereof as compared to the        non-isotopically enriched compound;    -   d) increased average plasma levels of at least one metabolite of        the compound per dosage unit thereof as compared to the        non-isotopically enriched compound; and    -   e) an improved clinical effect during the treatment in the        subject per dosage unit thereof as compared to the        non-isotopically enriched compound.

In certain embodiments, the method affects a decreased metabolism of thecompound per dosage unit thereof by at least onepolymorphically-expressed cytochrome P450 isoform in the subject, ascompared to the corresponding non-isotopically enriched compound.

In certain embodiments, the cytochrome P450 isoform is selected from thegroup consisting of CYP2C8, CYP2C9, CYP2C19, and CYP2D6.

In certain embodiments, the compound is characterized by decreasedinhibition of at least one cytochrome P450 or monoamine oxidase isoformin the subject per dosage unit thereof as compared to thenon-isotopically enriched compound.

In certain embodiments, the cytochrome P450 or monoamine oxidase isoformis selected from the group consisting of CYP1A1, CYP1A2, CYP1B1, CYP2A6,CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1,CYP2G1, CYP2J2, CYP2R1, CYP2S1, CYP3A4, CYP3A5, CYP3A5P1, CYP3A5P2,CYP3A7, CYP4A11, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12,CYP4X1, CYP4Z1, CYP5A1, CYP7A1, CYP7B1, CYP8A1, CYP8B1, CYP11A1,CYP11B1, CYP11B2, CYP17, CYP19, CYP21, CYP24, CYP26A1, CYP26B1, CYP27A1,CYP27B1, CYP39, CYP46, CYP51, MAO_(A), and MAO_(B).

In certain embodiments, the method reduces a deleterious change in adiagnostic hepatobiliary function endpoint, as compared to thecorresponding non-isotopically enriched compound.

In certain embodiments, the diagnostic hepatobiliary function endpointis selected from the group consisting of alanine aminotransferase(“ALT”), serum glutamic-pyruvic transaminase (“SGPT”), aspartateaminotransferase (“AST,” “SGOT”), ALT/AST ratios, serum aldolase,alkaline phosphatase (“ALP”), ammonia levels, bilirubin, gamma-glutamyltranspeptidase (“GGTP,” “γ-GTP,” “GGT”), leucine aminopeptidase (“LAP”),liver biopsy, liver ultrasonography, liver nuclear scan,5′-nucleotidase, and blood protein.

Also provided is a compound, or a salt thereof, as disclosed herein foruse as a medicament.

Also provided is a compound, or a salt thereof, as disclosed herein foruse in the manufacture of a medicament for the prevention or treatmentof a cystic fibrosis transmembrane conductance regulator-mediateddisorder.

All publications and references cited herein are expressly incorporatedherein by reference in their entirety. However, with respect to anysimilar or identical terms found in both the incorporated publicationsor references and those explicitly put forth or defined in thisdocument, then those terms definitions or meanings explicitly put forthin this document shall control in all respects.

As used herein, the terms below have the meanings indicated.

The singular forms “a,” “an,” and “the” may refer to plural articlesunless specifically stated otherwise.

The term “about,” as used herein, is intended to qualify the numericalvalues which it modifies, denoting such a value as variable within amargin of error. When no particular margin of error, such as a standarddeviation to a mean value given in a chart or table of data, is recited,the term “about” should be understood to mean that range which wouldencompass the recited value and the range which would be included byrounding up or down to that figure as well, taking into accountsignificant figures.

When ranges of values are disclosed, and the notation “from n₁ . . . ton₂” or “n₁-n₂” is used, where n₁ and n₂ are the numbers, then unlessotherwise specified, this notation is intended to include the numbersthemselves and the range between them. This range may be integral orcontinuous between and including the end values.

The term “deuterium enrichment” refers to the percentage ofincorporation of deuterium at a given position in a molecule in theplace of hydrogen. For example, deuterium enrichment of 1% at a givenposition means that 1% of molecules in a given sample contain deuteriumat the specified position. Because the naturally occurring distributionof deuterium is about 0.0156%, deuterium enrichment at any position in acompound synthesized using non-enriched starting materials is about0.0156%. The deuterium enrichment can be determined using conventionalanalytical methods known to one of ordinary skill in the art, includingmass spectrometry and nuclear magnetic resonance spectroscopy.

The term “is/are deuterium,” when used to describe a given position in amolecule such as R₁-R₂₃ or the symbol “D”, when used to represent agiven position in a drawing of a molecular structure, means that thespecified position is enriched with deuterium above the naturallyoccurring distribution of deuterium. The same is true of the term“contains deuterium,” which is often used to refer to methyl groupswhich may be mono-, di- or trideuterated (e.g., such groups may be—CH₂D, —CD₂H, and —CD₃, wherein the each position denoted D is enrichedwith deuterium above the naturally occurring distribution of deuterium).In one embodiment deuterium enrichment is no less than about 1%, inanother no less than about 5%, in another no less than about 10%, inanother no less than about 20%, in another no less than about 50%, inanother no less than about 70%, in another no less than about 80%, inanother no less than about 90%, or in another no less than about 98% ofdeuterium at the specified position.

The term “isotopic enrichment” refers to the percentage of incorporationof a less prevalent isotope of an element at a given position in amolecule in the place of the more prevalent isotope of the element.

The term “non-isotopically enriched” refers to a molecule in which thepercentages of the various isotopes are substantially the same as thenaturally occurring percentages.

Asymmetric centers exist in the compounds disclosed herein. Thesecenters are designated by the symbols “R” or “S,” depending on theconfiguration of substituents around the chiral carbon atom. It shouldbe understood that the invention encompasses all stereochemical isomericforms, including diastereomeric, enantiomeric, and epimeric forms, aswell as d-isomers and l-isomers, and mixtures thereof. Individualstereoisomers of compounds can be prepared synthetically fromcommercially available starting materials which contain chiral centersor by preparation of mixtures of enantiomeric products followed byseparation such as conversion to a mixture of diastereomers followed byseparation or recrystallization, chromatographic techniques, directseparation of enantiomers on chiral chromatographic columns, or anyother appropriate method known in the art. Starting compounds ofparticular stereochemistry are either commercially available or can bemade and resolved by techniques known in the art. Additionally, thecompounds disclosed herein may exist as geometric isomers. The presentinvention includes all cis, trans, syn, anti, entgegen (E), and zusammen(Z) isomers as well as the appropriate mixtures thereof. Additionally,compounds may exist as tautomers; all tautomeric isomers are provided bythis invention. Additionally, the compounds disclosed herein can existin unsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. In general, the solvatedforms are considered equivalent to the unsolvated forms.

The term “bond” refers to a covalent linkage between two atoms, or twomoieties when the atoms joined by the bond are considered to be part oflarger substructure. A bond may be single, double, or triple unlessotherwise specified. A dashed line between two atoms in a drawing of amolecule indicates that an additional bond may be present or absent atthat position.

The term “disorder” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disease” and“condition” (as in medical condition), in that all reflect an abnormalcondition of the human or animal body or of one of its parts thatimpairs normal functioning, is typically manifested by distinguishingsigns and symptoms.

The terms “treat,” “treating,” and “treatment” are meant to includealleviating or abrogating a disorder or one or more of the symptomsassociated with a disorder; or alleviating or eradicating the cause(s)of the disorder itself. As used herein, reference to “treatment” of adisorder is intended to include prevention. The terms “prevent,”“preventing,” and “prevention” refer to a method of delaying orprecluding the onset of a disorder; and/or its attendant symptoms,barring a subject from acquiring a disorder or reducing a subject's riskof acquiring a disorder.

The term “therapeutically effective amount” refers to the amount of acompound that, when administered, is sufficient to prevent developmentof, or alleviate to some extent, one or more of the symptoms of thedisorder being treated. The term “therapeutically effective amount” alsorefers to the amount of a compound that is sufficient to elicit thebiological or medical response of a cell, tissue, system, animal, orhuman that is being sought by a researcher, veterinarian, medicaldoctor, or clinician.

The term “subject” refers to an animal, including, but not limited to, aprimate (e.g., human, monkey, chimpanzee, gorilla, and the like),rodents (e.g., rats, mice, gerbils, hamsters, ferrets, and the like),lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline,and the like. The terms “subject” and “patient” are used interchangeablyherein in reference, for example, to a mammalian subject, such as ahuman patient.

The term “combination therapy” means the administration of two or moretherapeutic agents to treat a therapeutic disorder described in thepresent disclosure. Such administration encompasses co-administration ofthese therapeutic agents in a substantially simultaneous manner, such asin a single capsule having a fixed ratio of active ingredients or inmultiple, separate capsules for each active ingredient. In addition,such administration also encompasses use of each type of therapeuticagent in a sequential manner. In either case, the treatment regimen willprovide beneficial effects of the drug combination in treating thedisorders described herein.

The term “cystic fibrosis transmembrane conductance regulator” or“cystic fibrosis transmembrane conductance regulator protein” refers toa cAMP/ATP-mediated anion channel that is expressed in a variety ofcells types, including absorptive and secretory epithelia cells, whereit regulates anion flux across the membrane, as well as the activity ofother ion channels and proteins. In epithelia cells, normal functioningof cystic fibrosis transmembrane conductance regulator is critical forthe maintenance of electrolyte transport throughout the body,

including respiratory and digestive tissue. A defect in this gene causesmutations in cystic fibrosis transmembrane conductance regulatorresulting in cystic fibrosis, the most common fatal genetic disease inhumans. Cystic fibrosis affects approximately one in every 2,500 infantsin the United States. Within the general United States population, up to10 million people carry a single copy of the defective gene withoutapparent ill effects. In contrast, individuals with two copies of thecystic fibrosis associated gene suffer from the debilitating and fataleffects of cystic fibrosis, including chronic lung disease. In patientswith cystic fibrosis, mutations in cystic fibrosis transmembraneconductance regulator endogenously expressed in respiratory epithelialead to reduced apical anion secretion causing an imbalance in ion andfluid transport. The resulting decrease in anion transport contributesto enhance mucus accumulation in the lung and the accompanying microbialinfections that ultimately cause death in cystic fibrosis patients. Inaddition to respiratory disease, cystic fibrosis patients typicallysuffer from gastrointestinal problems and pancreatic insufficiency that,if left untreated, results in death. In addition, the majority of maleswith cystic fibrosis are infertile and fertility is decreased amongfemales with cystic fibrosis.

The term “cystic fibrosis transmembrane conductance regulator-mediateddisorder,” refers to a disorder that is characterized by abnormal cysticfibrosis transmembrane conductance regulator activity or cystic fibrosistransmembrane conductance regulator activity that, when modulated, leadsto the amelioration of other abnormal biological processes. A cysticfibrosis transmembrane conductance regulator-mediated disorder may becompletely or partially mediated by modulating cystic fibrosistransmembrane conductance regulator. In particular, a cystic fibrosistransmembrane conductance regulator-mediated disorder is one in whichmodulation of cystic fibrosis transmembrane conductance regulatorresults in some effect on the underlying disorder e.g., administrationof a cystic fibrosis transmembrane conductance regulator modulatorresults in some improvement in at least some of the patients beingtreated.

A modulator may activate the activity of a cystic fibrosis transmembraneconductance regulator, may activate or inhibit the activity of a cysticfibrosis transmembrane conductance regulator depending on theconcentration of the compound exposed to the cystic fibrosistransmembrane conductance regulator, or may inhibit the activity of acystic fibrosis transmembrane conductance regulator. Such activation orinhibition may be contingent on the occurrence of a specific event, suchas activation of a signal transduction pathway, and/or may be manifestonly in particular cell types. The term “cystic fibrosis transmembraneconductance regulator modulator” or “modulation of cystic fibrosistransmembrane conductance regulator” also refers to altering thefunction of a cystic fibrosis transmembrane conductance regulator byincreasing or decreasing the probability that a complex forms between ancystic fibrosis transmembrane conductance regulator and a naturalbinding partner. A cystic fibrosis transmembrane conductance regulatormodulator may increase the probability that such a complex forms betweenthe cystic fibrosis transmembrane conductance regulator and the naturalbinding partner, may increase or decrease the probability that a complexforms between the cystic fibrosis transmembrane conductance regulatorand the natural binding partner depending on the concentration of thecompound exposed to the cystic fibrosis transmembrane conductanceregulator, and or may decrease the probability that a complex formsbetween the cystic fibrosis transmembrane conductance regulator and thenatural binding partner. In some embodiments, modulation of the cysticfibrosis transmembrane conductance regulator may be assessed usingReceptor Selection and Amplification Technology (R-SAT) as described inWO 2014014841; WO 2013185112; WO 2012170061; WO 2011133956; WO2011133751; WO 2011119984; WO 2010054138; WO 2010053471; US 20130116238;US 20120046330; US 20120015999; and US 20090131492, the disclosure ofwhich is incorporated herein by reference in its entirety.

The term “therapeutically acceptable” refers to those compounds (orsalts, prodrugs, tautomers, zwitterionic forms, etc.) which are suitablefor use in contact with the tissues of patients without excessivetoxicity, irritation, allergic response, immunogenicity, arecommensurate with a reasonable benefit/risk ratio, and are effective fortheir intended use.

The term “pharmaceutically acceptable carrier,” “pharmaceuticallyacceptable excipient,” “physiologically acceptable carrier,” or“physiologically acceptable excipient” refers to apharmaceutically-acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, solvent, or encapsulatingmaterial. Each component must be “pharmaceutically acceptable” in thesense of being compatible with the other ingredients of a pharmaceuticalformulation. It must also be suitable for use in contact with the tissueor organ of humans and animals without excessive toxicity, irritation,allergic response, immunogenicity, or other problems or complications,commensurate with a reasonable benefit/risk ratio.

The terms “active ingredient,” “active compound,” and “active substance”refer to a compound, which is administered, alone or in combination withone or more pharmaceutically acceptable excipients or carriers, to asubject for treating, preventing, or ameliorating one or more symptomsof a disorder.

The terms “drug,” “therapeutic agent,” and “chemotherapeutic agent”refer to a compound, or a pharmaceutical composition thereof, which isadministered to a subject for treating, preventing, or ameliorating oneor more symptoms of a disorder.

The term “release controlling excipient” refers to an excipient whoseprimary function is to modify the duration or place of release of theactive substance from a dosage form as compared with a conventionalimmediate release dosage form.

The term “nonrelease controlling excipient” refers to an excipient whoseprimary function do not include modifying the duration or place ofrelease of the active substance from a dosage form as compared with aconventional immediate release dosage form.

The term “prodrug” refers to a compound functional derivative of thecompound as disclosed herein and is readily convertible into the parentcompound in vivo. Prodrugs are often useful because, in some situations,they may be easier to administer than the parent compound. They may, forinstance, be bioavailable by oral administration whereas the parentcompound is not. The prodrug may also have enhanced solubility inpharmaceutical compositions over the parent compound. A prodrug may beconverted into the parent drug by various mechanisms, includingenzymatic processes and metabolic hydrolysis.

The compounds disclosed herein can exist as therapeutically acceptablesalts. The term “therapeutically acceptable salt,” as used herein,represents salts or zwitterionic forms of the compounds disclosed hereinwhich are therapeutically acceptable as defined herein. The salts can beprepared during the final isolation and purification of the compounds orseparately by reacting the appropriate compound with a suitable acid orbase. Therapeutically acceptable salts include acid and basic additionsalts.

Suitable acids for use in the preparation of pharmaceutically acceptablesalts include, but are not limited to, acetic acid, 2,2-dichloroaceticacid, acylated amino acids, adipic acid, alginic acid, ascorbic acid,L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoicacid, boric acid, (+)-camphoric acid, camphorsulfonic acid,(+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylicacid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid,galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid,D-glucuronic acid, L-glutamic acid, α-oxo-glutaric acid, glycolic acid,hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid,(+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, lauric acid,maleic acid, (−)-L-malic acid, malonic acid, (±)-DL-mandelic acid,methanesulfonic acid, naphthalene-2-sulfonic acid,naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinicacid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid,pamoic acid, perchloric acid, phosphoric acid, L-pyroglutamic acid,saccharic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid,stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaricacid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, andvaleric acid.

Suitable bases for use in the preparation of pharmaceutically acceptablesalts, including, but not limited to, inorganic bases, such as magnesiumhydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, orsodium hydroxide; and organic bases, such as primary, secondary,tertiary, and quaternary, aliphatic and aromatic amines, includingL-arginine, benethamine, benzathine, choline, deanol, diethanolamine,diethylamine, dimethylamine, dipropylamine, diisopropylamine,2-(diethylamino)-ethanol, ethanolamine, ethylamine, ethylenediamine,isopropylamine, N-methyl-glucamine, hydrabamine, 1H-imidazole, L-lysine,morpholine, 4-(2-hydroxyethyl)-morpholine, methylamine, piperidine,piperazine, propylamine, pyrrolidine, 1-(2-hydroxyethyl)-pyrrolidine,pyridine, quinuclidine, quinoline, isoquinoline, secondary amines,triethanolamine, trimethylamine, triethylamine, N-methyl-D-glucamine,2-amino-2-(hydroxymethyl)-1,3-propanediol, and tromethamine.

While it may be possible for the compounds of the subject invention tobe administered as the raw chemical, it is also possible to present themas a pharmaceutical composition. Accordingly, provided herein arepharmaceutical compositions which comprise one or more of certaincompounds disclosed herein, or one or more pharmaceutically acceptablesalts, prodrugs, or solvates thereof, together with one or morepharmaceutically acceptable carriers thereof and optionally one or moreother therapeutic ingredients. Proper formulation is dependent upon theroute of administration chosen. Any of the well-known techniques,carriers, and excipients may be used as suitable and as understood inthe art; e.g., in Remington's Pharmaceutical Sciences. Thepharmaceutical compositions disclosed herein may be manufactured in anymanner known in the art, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or compression processes. The pharmaceuticalcompositions may also be formulated as a modified release dosage form,including delayed-, extended-, prolonged-, sustained-, pulsatile-,controlled-, accelerated- and fast-, targeted-, programmed-release, andgastric retention dosage forms. These dosage forms can be preparedaccording to conventional methods and techniques known to those skilledin the art.

The compositions include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous, intraarticular,and intramedullary), intraperitoneal, transmucosal, transdermal, rectaland topical (including dermal, buccal, sublingual and intraocular)administration although the most suitable route may depend upon forexample the condition and disorder of the recipient. The compositionsmay conveniently be presented in unit dosage form and may be prepared byany of the methods well known in the art of pharmacy. Typically, thesemethods include the step of bringing into association a compound of thesubject invention or a pharmaceutically salt, prodrug, or solvatethereof (“active ingredient”) with the carrier which constitutes one ormore accessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both and then,if necessary, shaping the product into the desired formulation.

Formulations of the compounds disclosed herein suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

Pharmaceutical preparations which can be used orally include tablets,push-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. Tablets maybe made by compression or molding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such as apowder or granules, optionally mixed with binders, inert diluents, orlubricating, surface active or dispersing agents. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein. All formulationsfor oral administration should be in dosages suitable for suchadministration. The push-fit capsules can contain the active ingredientsin admixture with filler such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds may be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added.Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. The formulations may be presentedin unit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in powder form or in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example, saline or sterile pyrogen-free water,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Formulations for parenteral administration include aqueous andnon-aqueous (oily) sterile injection solutions of the active compoundswhich may contain antioxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

For buccal or sublingual administration, the compositions may take theform of tablets, lozenges, pastilles, or gels formulated in conventionalmanner. Such compositions may comprise the active ingredient in aflavored basis such as sucrose and acacia or tragacanth.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter, polyethylene glycol, or otherglycerides.

Certain compounds disclosed herein may be administered topically, thatis by non-systemic administration. This includes the application of acompound disclosed herein externally to the epidermis or the buccalcavity and the instillation of such a compound into the ear, eye andnose, such that the compound does not significantly enter the bloodstream. In contrast, systemic administration refers to oral,intravenous, intraperitoneal and intramuscular administration.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of inflammation such as gels, liniments, lotions, creams,ointments or pastes, and drops suitable for administration to the eye,ear or nose.

For administration by inhalation, compounds may be delivered from aninsufflator, nebulizer pressurized packs or other convenient means ofdelivering an aerosol spray. Pressurized packs may comprise a suitablepropellant such as dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Alternatively, foradministration by inhalation or insufflation, the compounds according tothe invention may take the form of a dry powder composition, for examplea powder mix of the compound and a suitable powder base such as lactoseor starch. The powder composition may be presented in unit dosage form,in for example, capsules, cartridges, gelatin or blister packs fromwhich the powder may be administered with the aid of an inhalator orinsufflator.

Preferred unit dosage formulations are those containing an effectivedose, as herein below recited, or an appropriate fraction thereof, ofthe active ingredient.

Compounds may be administered orally or via injection at a dose of from0.1 to 500 mg/kg per day. The dose range for adult humans is generallyfrom 5 mg to 2 g/day. Tablets or other forms of presentation provided indiscrete units may conveniently contain an amount of one or morecompounds which is effective at such dosage or as a multiple of thesame, for instance, units containing 5 mg to 500 mg, usually around 10mg to 200 mg.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration.

The compounds can be administered in various modes, e.g. orally,topically, or by injection. The precise amount of compound administeredto a patient will be the responsibility of the attendant physician. Thespecific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diets, time ofadministration, route of administration, rate of excretion, drugcombination, the precise disorder being treated, and the severity of thedisorder being treated. Also, the route of administration may varydepending on the disorder and its severity.

In the case wherein the patient's condition does not improve, upon thedoctor's discretion the administration of the compounds may beadministered chronically, that is, for an extended period of time,including throughout the duration of the patient's life in order toameliorate or otherwise control or limit the symptoms of the patient'sdisorder.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the compounds may be given continuouslyor temporarily suspended for a certain length of time (i.e., a “drugholiday”).

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disorder is retained.Patients can, however, require intermittent treatment on a long-termbasis upon any recurrence of symptoms.

Disclosed herein are methods of treating a cystic fibrosis transmembraneconductance regulator-mediated disorder comprising administering to asubject having or suspected to have such a disorder, a therapeuticallyeffective amount of a compound as disclosed herein or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof.

Cystic fibrosis transmembrane conductance regulator-mediated disorders,include, but are not limited to, cystic fibrosis, sarcoglycanopathies,Brody's disease, cathecolaminergic polymorphic ventricular tachycardia,limb girdle muscular dystrophy, asthma, smoke induced chronicobstructive pulmonary disorder, chronic bronchitis, rhinosinusitis,constipation, pancreatitis, pancreatic insufficiency, male infertilitycaused by congenital bilateral absence of the vas deferens (CBAVD), mildpulmonary disease, idiopathic pancreatitis, allergic bronchopulmonaryaspergillosis (ABPA), liver disease, hereditary emphysema, hereditaryhemochromatosis, coagulation-fibrinolysis deficiencies, such as proteinC deficiency, type 1 hereditary angioedema, lipid processingdeficiencies, such as familial hypercholesterolemia, type 1chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, suchas I-cell disease/pseudo-Hurler, mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,polyendocrinopathy/hyperinsulinemia, diabetes mellitus, Laron dwarfism,myeloperoxidase deficiency, primary hypoparathyroidism, melanoma,glycanosis CDG type 1, congenital hyperthyroidism, osteogenesisimperfecta, hereditary hypofibrinogenemia, ACT deficiency, diabetesinsipidus (DI), neurohypophyseal DI, nephrogenic DI, Charcot-Marie toothsyndrome, Pelizaeus-Merzbacher disease, neurodegenerative diseases suchas Alzheimer's disease, Parkinson's disease, amyotrophic lateralsclerosis, progressive supranuclear palsy, Pick's disease, polyglutamineneurological disorders such as Huntington's, spinocerebellar ataxia typeI, spinal and bulbar muscular atrophy, dentatombral pallidoluysian, andmyotonic dystrophy, as well as spongifiorm encephalopathies, such ashereditary Creutzfeldt-Jakob disease (due to prion protein processingdefect), Fabry disease, Gerstrnarm-Straussler-Scheinker syndrome,chronic obstructive pulmonary disorder, dry-eye disease, or Sjogren'sdisease, osteoporosis, osteopenia, bone healing and bone growth(including bone repair, bone regeneration, reducing bone resorption andincreasing bone deposition), Gorham's Syndrome, chloride channelopathiessuch as myotonia congenita (Thomson and Becker forms), Bartter'ssyndrome type III, Dent's disease, hyperekplexia, epilepsy, lysosomalstorage disease, Angelman syndrome, and primary ciliary dyskinesia(PCD), a term for inherited disorders of the structure and/or functionof cilia, including PCD with situs inversus (also known as Kartagenersyndrome), PCD without situs inversus, and ciliary aplasia, and/or anydisorder which can lessened, alleviated, or prevented by administering acystic fibrosis transmembrane conductance regulator modulator.

In certain embodiments, a method of treating a cystic fibrosistransmembrane conductance regulator-mediated disorder comprisesadministering to the subject a therapeutically effective amount of acompound of as disclosed herein, or a pharmaceutically acceptable salt,solvate, or prodrug thereof, so as to affect: (1) decreasedinter-individual variation in plasma levels of the compound or ametabolite thereof; (2) increased average plasma levels of the compoundor decreased average plasma levels of at least one metabolite of thecompound per dosage unit; (3) decreased inhibition of, and/or metabolismby at least one cytochrome P450 or monoamine oxidase isoform in thesubject; (4) decreased metabolism via at least onepolymorphically-expressed cytochrome P450 isoform in the subject; (5) atleast one statistically-significantly improved disorder-control and/ordisorder-eradication endpoint; (6) an improved clinical effect duringthe treatment of the disorder, (7) prevention of recurrence, or delay ofdecline or appearance, of abnormal alimentary or hepatic parameters asthe primary clinical benefit, or (8) reduction or elimination ofdeleterious changes in any diagnostic hepatobiliary function endpoints,as compared to the corresponding non-isotopically enriched compound.

In certain embodiments, inter-individual variation in plasma levels ofthe compounds as disclosed herein, or metabolites thereof, is decreased;average plasma levels of the compound as disclosed herein are increased;average plasma levels of a metabolite of the compound as disclosedherein are decreased; inhibition of a cytochrome P450 or monoamineoxidase isoform by a compound as disclosed herein is decreased; ormetabolism of the compound as disclosed herein by at least onepolymorphically-expressed cytochrome P450 isoform is decreased; bygreater than about 5%, greater than about 10%, greater than about 20%,greater than about 30%, greater than about 40%, or by greater than about50% as compared to the corresponding non-isotopically enriched compound.

Plasma levels of the compound as disclosed herein, or metabolitesthereof, may be measured using methods known in the art.

Examples of cytochrome P450 isoforms in a mammalian subject include, butare not limited to, CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A13, CYP2B6,CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2G1, CYP2J2,CYP2R₁, CYP2S1, CYP3A4, CYP3A5, CYP3A5P1, CYP3A5P2, CYP3A7, CYP4A11,CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4X1, CYP4Z1,CYP5A1, CYP7A1, CYP7B1, CYP8A1, CYP8B1, CYP11A1, CYP11B1, CYP11B2,CYP17, CYP19, CYP21, CYP24, CYP26A1, CYP26B1, CYP27A1, CYP27B1, CYP39,CYP46, and CYP51.

Examples of monoamine oxidase isoforms in a mammalian subject include,but are not limited to, MAO_(A), and MAO_(B).

The inhibition of the cytochrome P450 isoform is measured by the methodof Ko et al. (British Journal of Clinical Pharmacology, 2000, 49,343-351). The inhibition of the MAO_(A) isoform is measured by themethod of Weyler et al. (J. Biol Chem. 1985, 260, 13199-13207). Theinhibition of the MAO_(B) isoform is measured by the method of Uebelhacket al. (Pharmacopsychiatry, 1998, 31, 187-192).

Examples of polymorphically-expressed cytochrome P450 isoforms in amammalian subject include, but are not limited to, CYP2C8, CYP2C9,CYP2C19, and CYP2D6.

The metabolic activities of liver microsomes, cytochrome P450 isoforms,and monoamine oxidase isoforms are measured by the methods describedherein.

Examples of improved disorder-control and/or disorder-eradicationendpoints, or improved clinical effects include, but are not limited to,change in sweat chloride, change in percent predicted forced expiratoryvolume in 1 second, change in forced expiratory volume in 1 second, andchange in cystic fibrosis questionnaire-revised (CFQ-R) respiratorydomain score.

Examples of diagnostic hepatobiliary function endpoints include, but arenot limited to, alanine aminotransferase (“ALT”), serum glutamic-pyruvictransaminase (“SGPT”), aspartate aminotransferase (“AST” or “SGOT”),ALT/AST ratios, serum aldolase, alkaline phosphatase (“ALP”), ammonialevels, bilirubin, gamma-glutamyl transpeptidase (“GGTP,” “γ-GTP,” or“GGT”), leucine aminopeptidase (“LAP”), liver biopsy, liverultrasonography, liver nuclear scan, 5′-nucleotidase, and blood protein.Hepatobiliary endpoints are compared to the stated normal levels asgiven in “Diagnostic and Laboratory Test Reference”, 4^(th) edition,Mosby, 1999. These assays are run by accredited laboratories accordingto standard protocol.

Besides being useful for human treatment, certain compounds andformulations disclosed herein may also be useful for veterinarytreatment of companion animals, exotic animals and farm animals,including mammals, rodents, and the like. More preferred animals includehorses, dogs, and cats.

Combination Therapy

The compounds disclosed herein may also be combined or used incombination with other agents useful in the treatment of cystic fibrosistransmembrane conductance regulator-mediated disorders. Or, by way ofexample only, the therapeutic effectiveness of one of the compoundsdescribed herein may be enhanced by administration of an adjuvant (i.e.,by itself the adjuvant may only have minimal therapeutic benefit, but incombination with another therapeutic agent, the overall therapeuticbenefit to the patient is enhanced).

Such other agents, adjuvants, or drugs, may be administered, by a routeand in an amount commonly used therefor, simultaneously or sequentiallywith a compound as disclosed herein. When a compound as disclosed hereinis used contemporaneously with one or more other drugs, a pharmaceuticalcomposition containing such other drugs in addition to the compounddisclosed herein may be utilized, but is not required.

In certain embodiments, the compounds disclosed herein can be combinedwith one or more antibiotics, bronchodilators, anticholinergics, DNase,mucolytics, nonsteroidal anti-inflammatory drugs, mast cell stabilizers,corticosteroids, or enzyme replacements.

In certain embodiments, the compounds disclosed herein can be combinedwith one or more antibiotic selected from the group consisting ofamikacin, amoxicillin, ampicillin, arsphenamine, azithromycin,aztreonam, azlocillin, bacitracin, carbenicillin, cefaclor, cefadroxil,cefamandole, cefazolin, cephalexin, cefdinir, cefditorin, cefepime,cefixime, cefoperazone, cefotaxime, cefoxitin, cefpodoxime, cefprozil,ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime,chloramphenicol, cilastin, ciprofloxacin, clarithromycin, clindamycin,cloxacillin, colistin, dalfopristan, demeclocycline, dicloxacillin,dirithromycin, doxycycline, erythromycin, enafloxacin, ertepenem,ethambutol, flucloxacillin, fosfomycin, furazolidone, gatifloxacin,geldanamycin, gentamicin, herbimicin, imipenem, isoniazide, kanamicin,levofloxacin, linezolid, lomefloxacin, loracarbef, mafenide,moxifloxacin, meropenem, metronidazole, mezlocillin, minocycline,mupirozin, nafcillin, neomycin, netilmicin, nitrofurantoin, norfloxacin,ofloxacin, oxytetracycline, penicillin, piperacillin, platensimycin,polymixin B, prontocil, pyrazinamide, quinupristine, retapamulin,rifampin, roxithromycin, spectinomycin, streptomycin, sulfacetamide,sulfamethizole, sulfamethoxazole, teicoplanin, telithromycin,tetracycline, ticarcillin, tobramycin, trimethoprim, troleandomycin,trovafloxacin, and vancomycin.

In certain embodiments, the compounds disclosed herein can be combinedwith one or more bronchodilator selected from the group consisting ofsalbutamol, levosalbutamol, terbutaline, pirbuterol, procaterol,metaproterenol, fenoterol, bitolterol mesylate, reproterol, salmeterol,formoterol, bambuterol, clenbuterol, and indacaterol.

In certain embodiments, the compounds disclosed herein can be combinedwith one or more anticholinergic selected from the group consisting ofoxyphencyclimine, camylofin, mebeverine, trimebutine, rociverine,dicycloverine, dihexyverine, difemerine, piperidolate, benzilone,glycopyrronium, oxyphenonium, penthienate, propantheline, otiloniumbromide, methantheline, tridihexethyl, isopropamide, hexocyclium,poldine, mepenzolate, bevonium, pipenzolate, biphemanil,(2-benzhydryloxyethyl)diethyl-methylammonium iodide, tiemonium iodide,prifinium bromide, timepidium bromide, tiotropium bromide, ipratropiumbromide, and fenpiverinium.

In certain embodiments, the compounds disclosed herein can be combinedwith one or more DNase selected from the group consisting of DNase Ienzyme, pulmozyme, and dornase alfa.

In certain embodiments, the compounds disclosed herein can be combinedwith one or more mucolytic selected from the group consisting ofacetylcysteine, ambroxol, carbocisteine, erdosteine, and mecysteine.

In certain embodiments, the compounds disclosed herein can be combinedwith one or more nonsteroidal anti-inflammatory drug selected from thegroup consisting of lumiracoxib, aceclofenac, acemetacin, amoxiprin,aspirin, azapropazone, benorilate, bromfenac, carprofen, celecoxib,choline magnesium salicylate, diclofenac, diflunisal, etodolac,etoracoxib, faislamine, fenbuten, fenoprofen, flurbiprofen, ibuprofen,indometacin, ketoprofen, ketorolac, lornoxicam, loxoprofen, meloxicam,meclofenamic acid, mefenamic acid, meloxicam, metamizole, methylsalicylate, magnesium salicylate, nabumetone, naproxen, nimesulide,oxyphenbutazone, parecoxib, phenylbutazone, piroxicam, salicylsalicylate, sulindac, sulfinprazone, suprofen, tenoxicam, tiaprofenicacid, and tolmetin.

In certain embodiments, the compounds disclosed herein can be combinedwith one or more mast cell stabilizer selected from the group consistingof cromolyn sodium and nedocromil sodium.

In certain embodiments, the compounds disclosed herein can be combinedwith one or more corticosteroid selected from the group consisting ofprednisone, prednisolne, hydrocortisone, beclometasone, ciclesonide,budesonide, flunisolide, betamethasone, fluticasone, triamcinolone, andmometasone.

In certain embodiments, the compounds disclosed herein can be combinedwith one or more enzyme replacement selected from the group consistingof pancrelipase, lipase, protease, and amylase.

The compounds disclosed herein can also be administered in combinationwith other classes of compounds, including, but not limited to,norepinephrine reuptake inhibitors (NRIs) such as atomoxetine; dopaminereuptake inhibitors (DARIs), such as methylphenidate;serotonin-norepinephrine reuptake inhibitors (SNRIs), such asmilnacipran; sedatives, such as diazepham; norepinephrine-dopaminereuptake inhibitor (NDRIs), such as bupropion;serotonin-norepinephrine-dopamine-reuptake-inhibitors (SNDRIs), such asvenlafaxine; monoamine oxidase inhibitors, such as selegiline;hypothalamic phospholipids; endothelin converting enzyme (ECE)inhibitors, such as phosphoramidon; opioids, such as tramadol;thromboxane receptor antagonists, such as ifetroban; potassium channelopeners; thrombin inhibitors, such as hirudin; hypothalamicphospholipids; growth factor inhibitors, such as modulators of PDGFactivity; platelet activating factor (PAF) antagonists; anti-plateletagents, such as GPIIb/IIIa blockers (e.g., abdximab, eptifibatide, andtirofiban), P2Y(AC) antagonists (e.g., clopidogrel, ticlopidine andCS-747), and aspirin; anticoagulants, such as warfarin; low molecularweight heparins, such as enoxaparin; Factor VIIa Inhibitors and FactorXa Inhibitors; renin inhibitors; neutral endopeptidase (NEP) inhibitors;vasopepsidase inhibitors (dual NEP-ACE inhibitors), such as omapatrilatand gemopatrilat; HMG CoA reductase inhibitors, such as pravastatin,lovastatin, atorvastatin, simvastatin, NK-104 (a.k.a. itavastatin,nisvastatin, or nisbastatin), and ZD-4522 (also known as rosuvastatin,or atavastatin or visastatin); squalene synthetase inhibitors; fibrates;bile acid sequestrants, such as questran; niacin; anti-atheroscleroticagents, such as ACAT inhibitors; MTP Inhibitors; calcium channelblockers, such as amlodipine besylate; potassium channel activators;alpha-muscarinic agents; beta-muscarinic agents, such as carvedilol andmetoprolol; antiarrhythmic agents; diuretics, such as chlorothlazide,hydrochiorothiazide, flumethiazide, hydroflumethiazide,bendroflumethiazide, methylchlorothiazide, trichioromethiazide,polythiazide, benzothlazide, ethacrynic acid, tricrynafen,chlorthalidone, furosenilde, musolimine, bumetanide, triamterene,amiloride, and spironolactone; thrombolytic agents, such as tissueplasminogen activator (tPA), recombinant tPA, streptokinase, urokinase,prourokinase, and anisoylated plasminogen streptokinase activatorcomplex (APSAC); anti-diabetic agents, such as biguanides (e.g.metformin), glucosidase inhibitors (e.g., acarbose), insulins,meglitinides (e.g., repaglinide), sulfonylureas (e.g., glimepiride,glyburide, and glipizide), thiozolidinediones (e.g. troglitazone,rosiglitazone and pioglitazone), and PPAR-gamma agonists;mineralocorticoid receptor antagonists, such as spironolactone andeplerenone; growth hormone secretagogues; aP2 inhibitors;phosphodiesterase inhibitors, such as PDE III inhibitors (e.g.,cilostazol) and PDE V inhibitors (e.g., sildenafil, tadalafil,vardenafil); protein tyrosine kinase inhibitors; antiinflammatories;antiproliferatives, such as methotrexate, FK506 (tacrolimus, Prograf),mycophenolate mofetil; chemotherapeutic agents; immunosuppressants;anticancer agents and cytotoxic agents (e.g., alkylating agents, such asnitrogen mustards, alkyl sulfonates, nitrosoureas, ethylenimines, andtriazenes); antimetabolites, such as folate antagonists, purineanalogues, and pyrridine analogues; antibiotics, such as anthracyclines,bleomycins, mitomycin, dactinomycin, and plicamycin; enzymes, such asL-asparaginase; farnesyl-protein transferase inhibitors; hormonalagents, such as glucocorticoids (e.g., cortisone),estrogens/antiestrogens, androgens/antiandrogens, progestins, andluteinizing hormone-releasing hormone anatagonists, and octreotideacetate; microtubule-disruptor agents, such as ecteinascidins;microtubule-stablizing agents, such as pacitaxel, docetaxel, andepothilones A-F; plant-derived products, such as vinca alkaloids,epipodophyllotoxins, and taxanes; and topoisomerase inhibitors;prenyl-protein transferase inhibitors; and cyclosporins; steroids, suchas prednisone and dexamethasone; cytotoxic drugs, such as azathiprineand cyclophosphamide; TNF-alpha inhibitors, such as tenidap; anti-TNFantibodies or soluble TNF receptor, such as etanercept, rapamycin, andleflunimide; and cyclooxygenase-2 (COX-2) inhibitors, such as celecoxiband rofecoxib; and miscellaneous agents such as, hydroxyurea,procarbazine, mitotane, hexamethylmelamine, gold compounds, platinumcoordination complexes, such as cisplatin, satraplatin, and carboplatin.

Thus, in another aspect, certain embodiments provide methods fortreating cystic fibrosis transmembrane conductance regulator-mediateddisorders in a human or animal subject in need of such treatmentcomprising administering to the subject an amount of a compounddisclosed herein effective to reduce or prevent the disorder in thesubject, in combination with at least one additional agent for thetreatment of the disorder that is known in the art. In a related aspect,certain embodiments provide therapeutic compositions comprising at leastone compound disclosed herein in combination with one or more additionalagents for the treatment of cystic fibrosis transmembrane conductanceregulator-mediated disorders.

General Synthetic Methods for Preparing Compounds

Isotopic hydrogen can be introduced into a compound as disclosed hereinby synthetic techniques that employ deuterated reagents, wherebyincorporation rates are predetermined; and/or by exchange techniques,wherein incorporation rates are determined by equilibrium conditions,and may be highly variable depending on the reaction conditions.Synthetic techniques, where tritium or deuterium is directly andspecifically inserted by tritiated or deuterated reagents of knownisotopic content, may yield high tritium or deuterium abundance, but canbe limited by the chemistry required. Exchange techniques, on the otherhand, may yield lower tritium or deuterium incorporation, often with theisotope being distributed over many sites on the molecule.

The compounds as disclosed herein can be prepared by methods known toone of skill in the art and routine modifications thereof, and/orfollowing procedures similar to those described in the Example sectionherein and routine modifications thereof, and/or procedures found in WO2014014841; WO 2013185112; WO 2012170061; WO 2011133956; WO 2011133751;WO 2011119984; WO 2010054138; WO 2010053471; US 20130116238; US20120046330; US 20120015999; US 20090131492, which are herebyincorporated in their entirety, and references cited therein and routinemodifications thereof. Compounds as disclosed herein can also beprepared as shown in any of the following schemes and routinemodifications thereof.

The following schemes can be used to practice the present invention. Anyposition shown as hydrogen may optionally be replaced with deuterium.

Compound 1 is treated with an appropriate acid, such as hydrochloricacid, in an appropriate solvent, such as water, to give compound 2.Compound 2 is treated with an appropriate metallating agent, such asmagnesium metal, in an appropriate solvent, such as tetrahydrofuran, togive an intermediate Grignard reagent, which was reacted with compound 3in an appropriate solvent, such as tetrahydrofuran, to give compound 4.Compound 4 is treated with an appropriate base, such as potassiumhydroxide, in an appropriate solvent, such as methanol, to give compound5. Compound 6 is reacted with an appropriate brominating agent, such asN-bromo-succinimide, in an appropriate solvent, such as ethyl acetate,to give compound 7. Compound 7 is reacted with compound 8 in thepresence of an appropriate catalyst, such as zinc perchlorate, in anappropriate solvent, such as toluene, at an elevated temperature, togive compound 9. Compound 9 is the treated with an appropriate reducingagent, such as a combination of hydrogen gas and 5% platinum on carbon,in an appropriate solvent, such as isopropyl acetate, to give compound10. Compound 10 is reacted with compound 5 (as the tosylate salt) in thepresence of an appropriate catalyst, such as a combination of palladiumacetate and 1,4-bis(diphenylphosphino)butane, in the presence of anappropriate base, such as potassium carbonate, in an appropriatesolvent, such as acetonitrile, at an elevated temperature, to givecompound 11. Compound 11 is treated with an appropriate catalyst, suchas diacetonitrile palladium dichloride, in an appropriate solvent, suchas acetonitrile, at an elevated temperature, to give compound 12.Compound 13 is treated with an appropriate reducing agent, such assodium bis(2-methoxyethoxy)aluminum hydride, in an appropriate solvent,such as toluene, to give compound 14. Compound 14 is treated with anappropriate chlorinating agent, such as thionyl chloride, in thepresence of an appropriate base, such as 4-dimethylamino pyridine, in anappropriate solvent, such as methyl tert-butyl ether, at a reducedtemperature, to give compound 15. Compound 15 is reacted with anappropriate cyanide salt, such as sodium cyanide, in an appropriatesolvent, such as dimethyl sulfoxide, to give compound 16. Compound 16 isreacted with compound 17 in the presence of an appropriate catalyst,such as tetrabutylammonium bromide, in the presence of an appropriatebase, such as sodium hydroxide, in an appropriate solvent, such as acombination of water and methyl tert-butyl ether, to give compound 18.Compound 18 is reacted with an appropriate base, such as sodiumhydroxide, in an appropriate solvent, such as ethanol, at an elevatedtemperature, to give compound 19. Compound 19 is reacted with anappropriate chlorinating agent, such as thionyl chloride, in anappropriate solvent, such as toluene, at an elevated temperature, togive an intermediate acid chloride which is then reacted with compound12 in the presence of an appropriate base, such as triethylamine, in anappropriate solvent, such as dichloromethane, to give compound 20.Compound 20 is the treated with an appropriate reducing agent, such as acombination of hydrogen gas and 5% palladium on carbon, in anappropriate solvent, such as tetrahydrofuran, to give a compound offormula I.

Deuterium can be incorporated to different positions synthetically,according to the synthetic procedures as shown in Scheme I, by usingappropriate deuterated intermediates. For example, to introducedeuterium at R₄-R₅, compound 1 with the corresponding deuteriumsubstitutions can be used. To introduce deuterium at one or morepositions of R₂-R₃, compound 2 with the corresponding deuteriumsubstitutions can be used. To introduce deuterium at one or morepositions of R₁₄-R₁₅ compound 6 with the corresponding deuteriumsubstitutions can be used. To introduce deuterium at R₇-R₈, and/orR₁₀-R₁₂ compound 8 with the corresponding deuterium substitutions can beused. To introduce deuterium at R₁₃, acetonitrile with the correspondingdeuterium substitutions can be used. To introduce deuterium at one ormore positions of R₂₁-R₂₃, compound 13 with the corresponding deuteriumsubstitutions can be used. To introduce deuterium at one or morepositions of R₁₇-R₂₀, compound 17 with the corresponding deuteriumsubstitutions can be used.

Deuterium can be incorporated to various positions having anexchangeable proton, such as the amine N—H and hydroxyl O—Hs, viaproton-deuterium equilibrium exchange. For example, to introducedeuterium at R₁, R₆, R₉, and R₁₆, these protons may be replaced withdeuterium selectively or non-selectively through a proton-deuteriumexchange method known in the art.

The invention is further illustrated by the following examples. AllIUPAC names were generated using CambridgeSoft's ChemDraw 10.0.

EXAMPLE 1(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide(VX-661)

Methyl 2,2-difluorobenzo[d][1,3]dioxole-5-carboxylate

To a 200 mL pressure tank reactor (10 atm. in CO), was placed5-bromo-2,2-difluoro-2H-1,3-benzodioxole (20.0 g, 84.4 mmol, 1.00equiv), methanol (40 mL), triethylamine (42.6 g, 5.00 equiv.), Pd₂(dba)₃(1.74 g, 1.69 mmol, 0.02 equiv), Pd(dppf)Cl₂ (1.4 g, 1.69 mmol, 0.02equiv.). The resulting solution was stirred at 85° C. under anatmosphere of CO overnight and the reaction progress was monitored byGCMS. The reaction mixture was cooled. The solids were filtered out. Theorganic phase was concentrated under vacuum to afford 17.5 g of methyl2,2-difluoro-2H-1,3-benzodioxole-5-carboxylate as a crude solid, whichwas used directly in the next step.

(2,2-difluorobenzo[d][1,3]dioxol-5-yl)methanol

To a 500 mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen were placed methyl2,2-difluoro-2H-1,3-benzodioxole-5-carboxylate (17.5 g, 81.01 mmol, 1.00equiv.), tetrahydrofuran (200 mL). This was followed by the addition ofLiAlH4 (6.81 mg, 162.02 mmol, 2.00 equiv.) at 0° C. The resultingsolution was stirred for 1 h at 25° C. and monitored by GCMS. Thereaction mixture was cooled to 0° C. until GCMS indicated the completionof the reaction. The pH value of the solution was adjusted to 8 withsodium hydroxide (1 mol/L). The solids were filtered out. The organiclayer combined and concentrated under vacuum to afford 13.25 g (87%) of(2,2-difluoro-2H-1,3-benzodioxol-5-yl)methanol as yellow oil.

5-(chloromethyl)-2,2-difluorobenzo[d][1,3]dioxole

(2,2-difluoro-2H-1,3-benzodioxol-5-yl)methanol (13.25 g, 70.4 mmol, 1.00equiv.) was dissolved in DCM (200 mL). Thionyl chloride (10.02 g, 1.20equiv.) was added to this solution. The resulting mixture was stirred atroom temperature for 4 hours and then concentrated under vacuum. Theresidue was then diluted with DCM (500 mL) and washed with 2×200 mL ofsodium bicarbonate and 1×200 mL of brine. The mixture was dried overanhydrous sodium sulfate, filtered and evaporated to afford 12.36 g(85%) of 5-(chloromethyl)-2,2-difluoro-2H-1,3-benzodioxole as yellowoil.

2-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)acetonitrile

5-(chloromethyl)-2,2-difluoro-2H-1,3-benzodioxole (12.36 g, 60 mmol,1.00 equiv.) was dissolved in DMSO (120 mL). This was followed by theaddition of NaCN (4.41 g, 1.50 equiv.) with the inert temperature below40° C. The resulting solution was stirred for 2 hours at roomtemperature. The reaction progress was monitored by GCMS. The reactionwas then quenched by the addition of 300 mL of water/ice. The resultingsolution was extracted with 3×100 mL of ethyl acetate. The organiclayers combined and washed with 3×100 mL brine dried over anhydroussodium sulfate and concentrated under vacuum to afford 10.84 g (92%) of2-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)acetonitrile as brown oil.

1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carbonitrile: Toa 100 mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, were placed2-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)acetonitrile (10.84 g, 55 mmol,1.00 equiv.), NaOH (50% in water), 1-bromo-2-chloroethane (11.92 g, 82.5mmol, 1.50 equiv.), Bu₄NBr (361 mg, 1.1 mmol, 0.02 equiv.). Theresulting solution was stirred for 48 h at 70° C. The reaction progresswas monitored by GCMS. The reaction mixture was cooled. The resultingsolution was extracted with 3×200 mL of ethyl acetate and the organiclayers combined. The resulting mixture was washed with 1×200 mL ofbrine. The mixture was dried over anhydrous sodium sulfate andconcentrated under vacuum to afford 10.12 g of1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carbonitrile asbrown oil.

1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxylic acid:To a 250-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carbonitrile(10.12 g, 45.38 mmol, 1.00 equiv), 6 N NaOH (61 mL) and EtOH (60 mL).The resulting solution was stirred for 3 h at 100° C. The reactionmixture was cooled and the pH value of the solution was adjusted to 2with hydrogen chloride (1 mol/L) until LCMS indicated the completion ofthe reaction. The solids were collected by filtration to afford 9.68 g(88%) of1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxylic acidas a light yellow solid.

1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carbonyl chloride

To a solution of1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxylic acid(687 mg, 2.84 mmol, 1.00 equiv.) in toluene (5 mL) was added thionylchloride (1.67 g, 5.00 equiv.). The resulting solution was stirred for 3h at 65° C. The reaction mixture was cooled and concentrated undervacuum to afford 738 mg (99%) of1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carbonyl chlorideas a yellow solid.

2-methyl-4-(trimethylsilyl)but-3-yn-2-ol

To a solution of ethynyltrimethylsilane (20 g, 203.63 mmol, 1.00 equiv)in THF (100 mL) was added n-BuLi (81 mL, 2.5M in THF) dropwise withstirring at −78° C. Then the resulting mixture was warmed to 0° C. for 1h with stirring and then cooled to −78° C. Propan-2-one (11.6 g, 199.73mmol, 1.00 equiv.) was added dropwise with the inert temperature below−78° C. The resulting solution was stirred at −78° C. for 3 h. Thereaction was then quenched by the addition of 100 mL of water andextracted with 3×100 mL of MTBE. The combined organic layers was driedover anhydrous sodium sulfate and concentrated under vacuum to afford 28g (90%) of 2-methyl-4-(trimethylsilyl)but-3-yn-2-ol as an off-whitesolid. ¹H NMR (400 MHz, CDCl₃) δ: 1.50 (s, 6H), 1.16-1.14 (m, 9H).

(3-chloro-3-methylbut-1-ynyl)trimethylsilane

To a 100 mL round-bottom flask, was placed 2-methyl-4-(trimethylsilyl)but-3-yn-2-ol (14 g, 89.57 mmol, 1.00 equiv.), conc. HCl (60 mL, 6.00equiv.). The resulting solution was stirred for 16 h at 0° C. Theresulting solution was extracted with 3×100 mL of hexane. The combinedorganic layers was dried over anhydrous sodium sulfate and concentratedunder vacuum to afford 8 g (51%) of(3-chloro-3-methylbut-1-yn-1-yl)trimethylsilane as light yellow oil. ¹HNMR (400 MHz, CDCl₃) (δ: 1.84 (s, 6H), 1.18-1.16 (m, 9H).

(4-(benzyloxy)-3,3-dimethylbut-1-ynyl)trimethylsilane

Magnesium turnings (1.32 g, 1.20 equiv) were charged to a 250-mL3-necked round-bottom flask and then suspended in THF (50 mL). Theresulting mixture was cooled to 0° C. and maintained with an inertatmosphere of nitrogen. (3-chloro-3-methylbut-1-yn-1-yl)trimethylsilane(8 g, 45.78 mmol, 1.00 equiv.) was dissolved in THF (50 mL) and thenadded dropwise to this mixture with the inert temperature between 33-37°C. The resulting solution was stirred at room temperature for anaddition 1 h before BnOCH₂Cl (6.45 g, 41.33 mmol, 0.90 equiv.) was addeddropwise with the temperature below 10° C. Then the resulting solutionwas stirred for 16 h at room temperature. The reaction was then quenchedby the addition of 50 mL of water and extracted with 3×100 mL of hexane.The combined organic layers was dried over anhydrous sodium sulfate andconcentrated under vacuum to afford 10 g (84%) of[4-(benzyloxy)-3,3-dimethylbut-1-yn-1-yl]trimethylsilane as light yellowoil. ¹H NMR (400 MHz, CDCl₃) (δ: 7.37-7.35 (m, 5H), 4.62 (s, 2H), 3.34(s, 2H), 1.24 (s, 6H), 0.17-0.14 (m, 9H).

((2,2-dimethylbut-3-ynyloxy)methyl)benzene

To a solution of[4-(benzyloxy)-3,3-dimethylbut-1-yn-1-yl]trimethylsilane (10 g, 38.40mmol, 1.00 equiv) in methanol (100 mL) was added potassium hydroxide(2.53 g, 38.33 mmol, 1.30 equiv). The resulting solution was stirred for16 h at room temperature. The resulting solution was diluted with 200 mLof water and extracted with 3×100 mL of hexane. The organic layerscombined and washed with 1×100 mL of water and then dried over anhydroussodium sulfate and concentrated under vacuum to afford 5 g (69%) of[[(2,2-dimethylbut-3-yn-1-yl)oxy]methyl]benzene as light yellow oil. ¹HNMR (300 MHz, D₂O) δ: 7.41-7.28 (m, 5H), 4.62 (s, 2H), 3.34 (s, 2H),2.14 (s, 1H), 1.32-1.23 (m, 9H).

methyl 2,2-difluorobenzo[d][1,3]dioxole-5-carboxylate

To a solution of 3-fluoro-4-nitroaniline (6.5 g, 41.64 mmol, 1.00 equiv)in chloroform (25 mL) and AcOH (80 mL) was added Bra (6.58 g, 41.17mmol, 1.00 equiv.) dropwise with stirring at 0° C. in 20 min. Theresulting solution was stirred for 2 h at room temperature. The reactionwas then quenched by the addition of 150 mL of water/ice. The pH valueof the solution was adjusted to 9 with sodium hydroxide (10%). Theresulting solution was extracted with 3×50 mL of ethyl acetate and theorganic layers combined. The resulting mixture was washed with 1×50 mLof water and 2×50 mL of brine, dried over anhydrous sodium sulfate andconcentrated under vacuum. The crude product was re-crystallized fromPE/EA (10:1) to afford 6 g (61%) of 2-bromo-5-fluoro-4-nitroaniline as ayellow solid.

(R)-1-(benzyloxy)-3-(2-bromo-5-fluoro-4-nitrophenylamino)propan-2-ol

2-bromo-5-fluoro-4-nitroaniline (6.00 g, 25.56 mmol, 1.00 equiv.),Zn(ClO₄)₂ (1.90 g, 5.1 mmol, 0.20 equiv.), 4A Molecular Sieves (3 g),toluene (60 mL) was stirred at room temperature for 2 h and maintainwith an inert atmosphere of N₂ until (2R)-2-[(benzyloxy)methyl]oxirane(1.37 g, 8.34 mmol, 2.00 equiv.) was added. Then the resulting mixturewas stirred for 15 h at 85° C. The reaction progress was monitored byLCMS. The solids were filtered out and the resulting solution wasdiluted with 20 mL of ethyl acetate. The resulting mixture was washedwith 2×20 mL of Sat. NH₄Cl and 1×20 mL of brine. The organic phase wasdried over anhydrous sodium sulfate and concentrated under vacuum. Theresidue was purified by a silica gel column, eluted with ethylacetate/petroleum ether (1:5) to afford 7.5 g (70%) ofN-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-bromo-5-fluoro-4-nitroanilineas a yellow solid.

(R)-1-(4-amino-2-bromo-5-fluorophenylamino)-3-(benzyloxy)propan-2-ol

To a 250-mL round-bottom flask, was placedN-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-bromo-5-fluoro-4-nitroaniline(7.5 g, 18.84 mmol, 1.00 equiv.), ethanol (80 mL), water (16 mL), NH₄Cl(10 g, 189 mmol, 10.00 equiv.), Zn (6.11 g, 18.84 mmol, 5.00 equiv.).The resulting solution was stirred for 4 h at 85° C. The solids werefiltered out and the resulting solution was concentrated under vacuumand diluted with 200 mL of ethyl acetate. The resulting mixture waswashed with 1×50 mL of water and 2×50 mL of brine. The organic phase wasdried over anhydrous sodium sulfate and concentrated under vacuum. Theresidue was purified by a silica gel column, eluted with ethylacetate/petroleum ether (1:3) to afford 4.16 g (60%) of1-N-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-bromo-5-fluorobenzene-1,4-diamineas light yellow oil.

(R)-4-(3-(benzyloxy)-2-hydroxypropylamino)-5-bromo-2-fluorobenzenaminium4-methylbenzenesulfonate

1-N-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-bromo-5-fluorobenzene-1,4-diamine(2 g, 5.42 mmol, 1.00 equiv.) was dissolved in dichloromethane (40 mL)followed by the addition of TsOH (1 g, 5.81 mmol, 1.10 equiv.). Theresulting mixture was stirred for 16 h at room temperature and thenconcentrated under vacuum to afford 2.8 g (95%) of4-[[(2R)-3-(benzyloxy)-2-hydroxypropyl]amino]-5-bromo-2-fluoroanilinium4-methylbenzene-1-sulfonate as an off-white solid.

(R)-1-(4-amino-2-(4-(benzyloxy)-3,3-dimethylbut-1-ynyl)-5-fluorophenylamino)3-(benzyloxy)propan-2-ol

To a 100-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed4-[[(2R)-3-(benzyloxy)-2-hydroxypropyl]amino]-5-bromo-2-fluoroanilinium4-methylbenzene-1-sulfonate (2.9 g, 5.36 mmol, 1.00 equiv.),[[(2,2-dimethylbut-3-yn-1-yl)oxy]methyl]benzene (1.2 g, 6.37 mmol, 1.20equiv.), Pd(OAc)₂ (48 mg, 0.21 mmol, 0.04 equiv.), dppb (138 mg, 0.32mmol, 0.06 equiv.), potassium carbonate (2.2 g, 15.92 mmol, 3.00 equiv.)and MeCN (50 mL). The resulting solution was stirred for 16 h at 80° C.The solids were filtered out and the resulting mixture was concentratedunder vacuum until LCMS indicated the completion of the reaction. Theresidue was purified by a silica gel column, eluted with ethylacetate/petroleum ether (1:4) to afford 2.2 g (86%) of1-N-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-[4-(benzyloxy)-3,3-dimethylbut-1-yn-1-yl]-5-fluorobenzene-1,4-diamineas a light brown solid.

1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxylic acid

To a 40-mL vial purged and maintained with an inert atmosphere ofnitrogen, was placed1-N-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-[4-(benzyloxy)-3,3-dimethylbut-1-yn-1-yl]-5-fluorobenzene-1,4-diamine(1 g, 2.1 mmol, 1.00 equiv.), MeCN (10 mL), Pd(MeCN)₂Cl₂ (82 mg, 0.32mmol, 0.15 equiv.). The resulting solution was stirred for 12 h at 85°C. The reaction progress was monitored by LCMS. The resulting mixturewas concentrated under vacuum to afford 900 mg (crude) of(2R)-1-[5-amino-2-[1-(benzyloxy)-2-methylpropan-2-yl]-6-fluoro-1H-indol-1-yl]-3-(benzyloxy)propan-2-olas a brown solid, which was used for next step without furtherpurification.

(R)—N-(1-(3-(benzyloxy)-2-hydroxypropyl)-2-(1-(benzyloxy)-2-methylpropan-2-yl)-6-fluoro-1H-indol-5-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamide

To a 40 mL vial purged and maintained with an inert atmosphere ofnitrogen, was placed(2R)-1-[5-amino-2-[1-(benzyloxy)-2-methylpropan-2-yl]-6-fluoro-1H-indol-1-yl]-3-(benzyloxy)propan-2-ol(800 mg, 1.68 mmol, 1.00 equiv.), dichloromethane (20 mL), TEA (508 mg,5.04 mmol, 3.00 equiv.).1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carbonyl chloride(524 mg, 2 mmol, 1.20 equiv.) was added to this mixture at 0° C. Theresulting solution was stirred for 2 h at 25° C. The reaction progresswas monitored by LCMS. The resulting solution was diluted with 20 mL ofDCM and washed with 3×10 mL of brine. The combined organic layers wasdried over anhydrous sodium sulfate and concentrated under vacuum. Theresidue was purified by a silica gel column, eluted with ethylacetate/petroleum ether (1:5) to afford 400 mg (30%) ofN-[1-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-[1-(benzyloxy)-2-methylpropan-2-yl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamideas a light yellow solid.

(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide

To a 100-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of H₂, were placedN-[1-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-[1-(benzyloxy)-2-methylpropan-2-yl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide(400 mg, 0.77 mmol, 1.00 equiv.) dry Pd/C (300 mg) and MeOH (5 Ml, 6MHCl). The resulting mixture was stirred at room temperature for 2 huntil LCMS indicated the completion of the reaction. The solids werefiltered out and the resulting mixture was concentrated under vacuum.The residue was purified by prep-HPLC with the following conditions:Column, XBridge Prep C18 OBD Column 19×150 mm, Sum; mobile phase andGradient, Phase A: Waters (0.1% FA), Phase B: ACN; Detector, UV 254 nmto afford 126.1 mg (42.4%) of(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamideas a light yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ: 8.32 (s, 1H), 7.54(s, 1H), 7.41-7.38 (m, 2H), 7.34-7.31 (m, 2H), 6.22 (s, 1H), 5.03-5.02(m, 1H), 4.93-4.90 (m, 1H), 4.77-4.75 (m, 1H), 4.42-4.39 (m, 1H),4.14-4.08 (m, 1H), 3.91 (brs, 1H), 3.64-3.57 (m, 2H), 3.47-3.40 (m, 2H),1.48-1.46 (m, 2H), 1.36-1.32 (m, 6H), 1.14-1.12 (m, 2H). LCMS:m/z=521.2[M+H]⁺.

EXAMPLE 21-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carbonyl chloride

Methyl 2,2-dimethyl-4-(trimethylsilyl)but-3-ynoate

To a 100-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed Mg (860 mg, 36 mmol, 1.20equiv), tetrahydrofuran (60 mL). And then(3-chloro-3-methylbut-1-yn-1-yl)trimethylsilane (5.22 g, 29.87 mmol,1.00 equiv.) was added dropwise to this mixture with the inerttemperature between 33-37° C. The resulting solution was stirred for anaddition 1 h at room temperature before methyl chloroformate (2.82 mg,29.84 mmol, 1.00 equiv.) was added at −78° C. The resulting solution wasthen stirred for 16 h at room temperature. The reaction was thenquenched by the addition of 60 mL of 1 M HCl and extracted with 2×60 mLof petroleum ether. The organic layers were combined, dried overanhydrous sodium sulfate and concentrated under vacuum to afford 4.7 g(79%) of methyl 2,2-dimethyl-4-(trimethylsilyl)but-3-ynoate as lightyellow oil. ¹H NMR (400 MHz, CDCl₃) δ: 3.76 (s, 3H), 1.48 (s, 6H),0.19-0.15 (m, 9H).

2-methyl-2-[2-(trimethylsilyl)ethynyl](1,1-²H₂)propan-1-ol

To a solution of methyl 2,2-dimethyl-4-(trimethylsilyl)but-3-ynoate (3g, 15.13 mmol, 1.00 equiv.) in tetrahydrofuran (50 mL) under an inertatmosphere of nitrogen was added LiAlD4 (760 mg, 1.10 equiv) at 0° C.The resulting solution was stirred for 1 h at 0° C. The reaction wasthen quenched by the addition of 50 mL of 1M HCl and extracted with 3×50mL of MTBE. The organic layers were combined, dried over anhydroussodium sulfate, filtered and concentrated under vacuum to afford 2.18 g(84%) of 2-methyl-2-[2-(trimethylsilyl)ethynyl](1,1-²H₂)propan-1-ol aslight yellow oil. ¹H NMR (400 MHz, CDCl₃) δ: 1.25-1.21 (m, 6H),0.18-0.15 (m, 9H).

([[2,2-dimethyl(1,1-²H₂)but-3-yn-1-yl]oxy]methyl)benzene

To a solution of2-methyl-2-[2-(trimethylsilyl)ethynyl](1,1-²H₂)propan-1-ol (2.1 g, 12.19mmol, 1.00 equiv.), tetrahydrofuran (50 mL) under an inert atmosphere ofnitrogen was added sodium hydride (730 mg, 18.25 mmol, 1.50 equiv) at 0°C. The resulting mixture was stirred for an addition 1 h. Then TBAI (450mg, 1.22 mmol, 0.10 equiv) and BnBr (1.9 g, 11.11 mmol, 0.90 equiv.)were added at 0° C. The resulting solution was stirred at roomtemperature for 24 h and then quenched by the addition of 50 mL ofwater. The resulting solution was extracted with 40 mL of petroleumether. The organic layers were combined, dried over anhydrous sodiumsulfate, filtered and concentrated under vacuum to afford 1.2 g (52%) of([[2,2-dimethyl(1,1-²H₂)but-3-yn-1-yl]oxy]methyl)benzene as light yellowoil. ¹H NMR (400 MHz, CDCl₃) δ: 7.40-7.30 (m, 5H), 4.64-4.60 (m, 2H),2.16 (s, 1H), 1.30-1.26 (m, 9H).

1-N-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-[4-(benzyloxy)-3,3-dimethyl(1,1-²H₂)but-1-yn-1-yl]-5-fluorobenzene-1,4-diamine

To a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed4-[[(2R)-3-(benzyloxy)-2-hydroxypropyl]amino]-5-bromo-2-fluoroanilinium4-methylbenzene-1-sulfonate (1.5 g, 2.77 mmol, 1.00 equiv),([2,2-dimethyl(1,1-²H₂)but-3-yn-1-yl]oxymethyl)benzene (790 mg, 4.15mmol, 1.50 equiv), potassium carbonate (1.15 g, 8.32 mmol, 3.00 equiv.),Pd(OAc)₂ (24.9 mg, 0.11 mmol, 0.04 equiv), dppb (70.9 mg, 0.17 mmol,0.06 equiv.), and MeCN (20 mL). The resulting solution was stirred for16 h at 80° C. The solids were filtered out. The organic layers werecombined, dried over anhydrous sodium sulfate, filtered and concentratedunder vacuum The residue was purified by a silica gel column, elutedwith ethyl acetate/petroleum ether (1:5) to afford 1 g (75%) of1-N-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-[4-(benzyloxy)-3,3-dimethyl(1,1-²H₂)but-1-yn-1-yl]-5-fluorobenzene-1,4-diamineas light yellow oil.

(2R)-1-[5-amino-2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-6-fluoro-1Hindol-1-yl]-3-(benzyloxy)propan-2-ol

To a 40-mL sealed tube purged and maintained with an inert atmosphere ofnitrogen, was placed1-N-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-[4-(benzyloxy)-3,3-dimethyl(1,1-²H₂)but-1-yn-1-yl]-5-fluorobenzene-1,4-diamine(1.0 g, 2.09 mmol, 1.00 equiv), MeCN (10 mL) and Pd(MeCN)₂Cl₂ (81.3 mg,0.31 mmol, 0.15 equiv). The resulting solution was stirred for 16 h at80° C. to afford 1.0 g of the desired product, which was used withoutfurther purification.

N-[1-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-5-fluoro-1H-indol-6-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide:To a 50-mL round-bottom flask, was placed(2R)-1-[5-amino-2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-6-fluoro-1H-indol-1-yl]-3-(benzyloxy)propan-2-ol(1.0 g, 2.09 mmol, 1.00 equiv), dichloromethane (20 mL),1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carbonyl chloride(750 mg, 2.88 mmol, 1.36 equiv.) and TEA (634 mg, 6.27 mmol, 3.00equiv.). The resulting solution was stirred for 1 h at 0° C. The residuewas purified by a silica gel column, eluted with ethyl acetate/petroleumether (1:5) to afford 500 mg (34%) ofN-[1-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-5-fluoro-1H-indol-6-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamideas light yellow oil.

1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carbonyl chloride

To a 100 mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of H₂, was placedN-[1-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-5-fluoro-1H-indol-6-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide(500 mg, 0.71 mmol, 1.00 equiv), dry Pd/C (300 mg) and MeOH (5 mL, 6 MHCl). The resulting mixture was stirred at room temperature for 2 huntil LCMS indicated the completion of the reaction. The solids werefiltered out and the organic phase was concentrated under vacuum. Theresidue was purified by prep-HPLC with the following conditions: Column,XBridge Prep C18 OBD Column 19×150 mm, Sum; mobile phase and Gradient,Phase A: Waters (0.1% FA), Phase B: ACN; Detector, UV 254 nm to afford148.2 mg (39.9%) of(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamideas a light yellow solid. ¹H NMR (300 MHz, DMSO-d6) δ: 8.32 (s, 1H), 7.53(s, 1H), 7.43-7.38 (m, 2H), 7.34-7.31 (m, 2H), 6.22 (s, 1H), 5.04-5.02(m, 1H), 4.93-4.91 (m, 1H), 4.72 (s, 1H), 4.42-4.39 (m, 1H), 4.14-4.08(m, 1H), 3.91 (brs, 1H), 3.48-3.40 (m, 2H), 1.48-1.47 (m, 2H), 1.35-1.24(m, 6H), 1.14-1.13 (m, 2H). LCMS: m/z=523.2 [M+H]+.

EXAMPLE 31-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-[1-hydroxy-2-methyl(²H₆)propan-2-yl]-1H-indol-5-yl]cyclopropane-1-carboxamide

2-[2-(trimethylsilyl)ethynyl](²H₆)propan-2-ol

To a 250-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed ethynyltrimethylsilane (9.8 g,99.78 mmol, 1.00 equiv.), tetrahydrofuran (100 mL) and cooled to −78° C.To this solution n-BuLi (40 mL, 2.5M in THF) was added dropwise at −78°C. The resulting mixture was then stirred at 0° C. for 1 h and thencooled to −78° C. Acetone-d₆ (6.4 g, 99.82 mmol, 1.00 equiv) was thenadded dropwise at −78° C. The resulting solution was stirred at roomtemperature for 3 h. and then quenched by the addition of 50 mL ofwater. The resulting solution was extracted with 2×50 mL of MTBE and theorganic layers were combined, dried over anhydrous sodium sulfate andconcentrated under vacuum to afford 14 g (86%) of2-[2-(trimethylsilyl)ethynyl](²H₆)propan-2-ol as an off-white solid.

[3-chloro-3-(²H₆)methyl(1,1-H)but-1-yn-1-yl]trimethylsilane

To a 250-mL round-bottom flask, was placed2-[2-(trimethylsilyl)ethynyl](²H₆)propan-2-ol (14 g, 86.24 mmol, 1.00equiv), hydrogen chloride (100 mL). The resulting solution was stirredfor 16 h at 20° C. The resulting solution was extracted with 2×50 mL ofMTBE and the organic layers were combined, dried over anhydrous sodiumsulfate and concentrated under vacuum to afford 7.5 g (48%) of[3-chloro-3-(²H₆)methyl(1,1-H)but-1-yn-1-yl]trimethylsilane as lightyellow oil.

([[2,2-bis(²H₆)-dimethylbut-3-yn-1-yl]oxy]methyl)benzene

Magnesium turnings (1.09 g, 45.42 mmol, 1.10 equiv.) were charged to a250-mL 3-necked round-bottom flask and then suspended in THF (20 mL).The resulting mixture was cooled to 0° C. and maintained with an inertatmosphere of nitrogen.[3-chloro-3-(²H₆)methyl(1,1-H)but-1-yn-1-yl]trimethylsilane (7.5 g,41.49 mmol, 1.00 equiv) was dissolved in THF (30 mL) and then addeddropwise to this mixture with the inert temperature between 33-37° C.The resulting solution was stirred at room temperature for an addition 1h before BnOCH₂Cl (5.83 g, 37.36 mmol, 0.90 equiv.) was added dropwisewith the temperature below 10° C. Then the resulting solution wasstirred for 16 h at room temperature. The reaction was then quenched bythe addition of 50 mL of water and extracted with 3×100 mL of hexane.The organic layers were combined, dried over anhydrous sodium sulfateand concentrated under vacuum to afford 10 g (84%) of([[2,2-bis-(²H₆)-dimethylbut-3-yn-1-yl]oxy]methyl)benzene as lightyellow oil.

([[2,2-bis(²H₆)methylbut-3-yn-1-yl]oxy]methyl)benzene

To a 250-mL round-bottom flask, was placed([[2,2-bis-²H₆-dimethylbut-3-yn-1-yl]oxy]methyl)benzene (10 g, 38.40mmol, 1.00 equiv.), potassium hydroxide (2.53 g, 38.33 mmol, 1.30equiv.), methanol (100 mL). The resulting solution was stirred for 16 hat room temperature. The resulting solution was diluted with 200 mL ofwater and extracted with 3×100 mL of hexane. The organic layers werecombined, washed with 1×100 mL of water, dried over anhydrous sodiumsulfate and concentrated under vacuum to afford 5 g (69%) of([[2,2-bis(²H₆)methylbut-3-yn-1-yl]oxy]methyl)benzene as light yellowoil.

1-N-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-[4-(benzyloxy)-3,3-bis(²H₆)methylbut-1-yn-1-yl]-5-fluorobenzene-1,4-diamine

To a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed4-[[(2R)-3-(benzyloxy)-2-hydroxypropyl]amino]-5-bromo-2-fluoroanilinium4-methylbenzene-1-sulfonate (1.5 g, 2.77 mmol, 1.00 equiv.),([[2,2-bis(²H₆)methylbut-3-yn-1-yl]oxy]methyl)benzene (815 mg, 4.15mmol, 1.00 equiv), potassium carbonate (1.15 g, 8.32 mmol, 3.00 equiv.),Pd(OAc)₂ (24.9 mg, 0.11 mmol, 0.04 equiv.), dppb (70.9 mg, 0.17 mmol,0.06 equiv.) and MeCN (20 mL). The resulting solution was stirred for 16h at 80° C. The solids were filtered out. The organic phase was driedover anhydrous sodium sulfate and concentrated under vacuum. The residuewas purified by a silica gel column, eluted with ethyl acetate/petroleumether (1:5) to afford 1 g (75%) of1-N-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-[4-(benzyloxy)-3,3-bis(²H₆)methylbut-1-yn-1-yl]-5-fluorobenzene-1,4-diamineas light yellow oil.

(2R)-1-[5-amino-2-[1-(benzyloxy)-2-bismethyl(²H₆)propan-2-yl]-6-fluoro-1H-indol-1-yl]-3-(benzyloxy)propan-2-ol

To a 40-mL sealed tube purged and maintained with an inert atmosphere ofnitrogen, was placed1-N-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-[4-(benzyloxy)-3,3-bis(²H₆)methylbut-1-yn-1-yl]-5-fluorobenzene-1,4-diamine(1.0 g, 2.09 mmol, 1.00 equiv.), MeCN (10 mL), Pd(MeCN)₂Cl₂ (81.3 mg,0.31 mmol, 0.15 equiv). The resulting solution was stirred for 16 h at80° C. to afford 1.0 g of the desired product, which was used withoutfurther purification.

N-[2-[1-(benzyloxy)-2-bismethyl(3,3-²H₆)propan-2-yl]-1-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide

To a 50-mL round-bottom flask, was placed(2R)-1-[5-amino-2-[1-(benzyloxy)-2-bismethyl(²H₆)propan-2-yl]-6-fluoro-1H-indol-1-yl]-3-(benzyloxy)propan-2-ol(1.0 g, 2.09 mmol, 1.00 equiv.), dichloromethane (20 mL),1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carbonyl chloride(750 mg, 2.88 mmol, 1.36 equiv.), TEA (634 mg, 6.27 mmol, 3.00 equiv.).The resulting solution was stirred for 1 h at 0° C. The residue waspurified by a silica gel column, eluted with ethyl acetate/petroleumether (1:5) to afford 500 mg (34%) ofN-[2-[1-(benzyloxy)-2-bismethyl(3,3-²H₆)propan-2-yl]-1-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamideas light yellow oil.

1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-[1-hydroxy-2-methyl(²H₆)propan-2-yl]-1H-indol-5-yl]cyclopropane-1-carboxamide

To a 100-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of H₂, was placedN-[2-[1-(benzyloxy)-2-bismethyl(3,3-²H₆)propan-2-yl]-1-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide(500 mg, 0.71 mmol, 1.00 equiv.) dry Pd/C (300 mg) and MeOH (6M HCl).The resulting mixture was stirred at room temperature for 2 h until LCMSindicated the completion of the reaction. The solids were filtered outand the resulting mixture was concentrated under vacuum. The residue waspurified by prep-HPLC with the following conditions: Column, XBridgePrep C18 OBD Column 19×150 mm, Sum; mobile phase and Gradient, Phase A:Waters (0.1% FA), Phase B: ACN; Detector, UV 254 nm to afford 116.1 mg(31.2%) of1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-[1-hydroxy-2-methyl(²H₆)propan-2-yl]-1H-indol-5-yl]cyclopropane-1-carboxamideas alight yellow solid. ¹H NMR (400 MHz, DMSO-d6) δ 8.31 (s, 1H), 7.53(s, 1H), 7.41-7.37 (m, 2H), 7.33-7.30 (m, 2H), 6.22 (s, 1H), 4.42-4.37(m, 1H), 4.12-4.06 (m, 1H), 3.90-3.89 (m, 1H), 3.62-3.54 (m, 2H),3.46-3.34 (m, 6H), 1.47-1.44 (m, 2H), 1.13-1.11 (m, 2H). LCMS:m/z=527.2[M+H]⁺.

EXAMPLE 4(R)1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)-N-[1-[2,3-dihydroxy(2,3,3-²H₃)propyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl]cyclopropane-1-carboxamide

oxiran-2-yl(²H₂)methanol

NaBD₄ (4.34 g, 103 mmol, 1.00 equiv.) was added to a solution of ethyloxirane-2-carboxylate (20 g, 172 mmol, 1.00 equiv.) in CD₃OD (300 mL) at−10° C. The resulting solution was stirred at −10° C. for 1 h. Thereaction was quenched by D₂O (3 mL) and evaporated with the temperaturebelow 40° C. The residue was extracted with 3×100 mL MTBE. The organiclayers were combined, dried over anhydrous sodium sulfate andconcentrated under vacuum to afford 10 g (76%) ofoxiran-2-yl(²H₂)methanol as off-white oil.

2-[(benzyloxy)(²H₂)methyl]oxirane

Oxiran-2-yl(²H₂)methanol (10 g, 131 mmol, 1.00 equiv) was dissolved inDMF (200 mL). To this solution was added NaH (6.32 g, 157 mmol, 1.20equiv) at 0° C. and the resulting mixture was stirred at 0° C. for anaddition 1 h. Then BnBr (22.5 g, 131 mmol, 1.00 equiv.) was addeddropwise to this reaction. The resulting solution was stirred for 2 h atroom temperature. The reaction was quenched by D20 and extracted with2×50 mL of EA. The organic layers were combined, dried over anhydroussodium sulfate and concentrated under vacuum. The residue was appliedonto a silica gel column with ethyl acetate/petroleum ether (1:5) toafford 15 g (69%) of 2-[(benzyloxy)(²H₂)methyl]oxirane as off-white oil.

N-[3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-bromo-5-fluoro-4-nitroaniline

2-bromo-5-fluoro-4-nitroaniline (6.00 g, 25.56 mmol, 1.00 equiv.),Zn(ClO₄)₂ (1.90 g, 5.1 mmol, 0.20 equiv.), 4A Molecular Sioves (3 g),toluene (60 mL) was stirred at room temperature for 2 h and maintainwith an inert atmosphere of N₂ until 2-[(benzyloxy)(²H₂)methyl]oxirane(1.37 g, 8.34 mmol, 2.00 equiv) was added. Then the resulting mixturewas stirred for 16 h at 85° C. The reaction progress was monitored byLCMS. The solids were filtered out and the resulting solution wasdiluted with 20 mL of ethyl acetate. The resulting mixture was washedwith 2×20 mL of Sat.NH₄Cl and 1×20 mL of brine. The organic layers werecombined, dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was applied onto a silica gel column with ethylacetate/petroleum ether (1:5) to afford 7.5 g (70%) ofN-[3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-bromo-5-fluoro-4-nitroanilineas a yellow solid.

1-N-[3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-bromo-5-fluorobenzene-1,4-diamine

To a 250-mL round-bottom flask, was placedN-[3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-bromo-5-fluoro-4-nitroaniline(7.5 g, 18.84 mmol, 1.00 equiv), ethanol (80 mL), water (16 mL), NH₄Cl(10 g, 189 mmol, 10.00 equiv.), Zn (6.11 g, 18.84 mmol, 5.00 equiv). Theresulting solution was stirred for 4 h at 85° C. The solids werefiltered out and the resulting solution was concentrated under vacuumand diluted with 200 mL of ethyl acetate. The resulting mixture waswashed with 1×50 mL of water and 2×50 mL of brine. The organic layerswere combined, dried over anhydrous sodium sulfate and concentratedunder vacuum. The residue was applied onto a silica gel column withethyl acetate/petroleum ether (1:3) to afford 4 g (58%) of1-N-[(2R)-3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-bromo-5-fluorobenzene-1,4-diamineas light yellow oil.

4-(3-(benzyloxy)-2-hydroxy(3,3-²H₂)propylamino)-5-bromo-2-fluorobenzenaminium4-methylbenzenesulfonate

1-N-[(2R)-3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-bromo-5-fluorobenzene-1,4-diamine(4 g, 10.84 mmol, 1.00 equiv.) was dissolved in dichloromethane (50 mL)followed by the addition of TsOH (2 g, 11.62 mmol, 1.10 equiv.). Theresulting mixture was stirred for 16 h at room temperature and thenconcentrated under vacuum to afford 5.6 g (95%) of4-(3-(benzyloxy)-2-hydroxy(3,3-²H₂)propylamino)-5-bromo-2-fluorobenzenaminium4-methylbenzenesulfonate as an off-white solid.

1-N-[3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-[4-(benzyloxy)-3,3-dimethylbut-1-yn-1-yl]-5-fluorobenzene-1,4-diamine

To a 250-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed4-(3-(benzyloxy)-2-hydroxy(3,3-²H₂)propylamino)-5-bromo-2-fluorobenzenaminium4-methylbenzenesulfonate (5.60 g, 10.33 mmol, 1.00 equiv.),([2,2-dimethylbut-3-yn-1-yl]oxymethyl)benzene (2.91 g, 15.50 mmol, 1.50equiv.), potassium carbonate (4.27 g, 30.10 mmol, 3.00 equiv.), Pd(OAc)₂(92.8 mg, 0.41 mmol, 0.04 equiv.), dppb (264.3 mg, 0.62 mmol, 0.06equiv.), MeCN (100 mL). The resulting solution was stirred for 16 h at80° C. The solids were filtered out. The organic phase was dried overanhydrous sodium sulfate and concentrated under vacuum. The residue wasapplied onto a silica gel column with ethyl acetate/petroleum ether(1:5) to afford 3.36 g (60%) of1-N-[3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-[4-(benzyloxy)-3,3-dimethylbut-1-yn-1-yl]-5-fluorobenzene-1,4-diamineas light yellow oil.

1-[5-amino-2-[1-(benzyloxy)-2-methylpropan-2-yl]-6-fluoro-1H-indol-1-yl]-3-(benzyloxy)(3,3-²H₂)propan-2-ol

To a 100-mL sealed tube purged and maintained with an inert atmosphereof nitrogen, was placed1-N-[3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-[4-(benzyloxy)-3,3-dimethylbut-1-yn-1-yl]-5-fluorobenzene-1,4-diamine(3.36 g, 7.01 mmol, 1.00 equiv.), MeCN (40 mL), Pd(MeCN)₂Cl₂ (273.2 mg,1.04 mmol, 0.15 equiv.). The resulting solution was stirred for 16 h at80° C. The solids were filtered and the filtrate was concentrated toafford 3.38 g of crude product, which was used without furtherpurification.

N-[1-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-5-fluoro-1H-indol-6-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide

To a 50-mL round-bottom flask, was placed1-[5-amino-2-[1-(benzyloxy)-2-methylpropan-2-yl]-6-fluoro-1H-indol-1-yl]-3-(benzyloxy)(3,3-²H₂)propan-2-ol(3.38 g, 7.01 mmol, 1.00 equiv.), dichloromethane (60 mL),1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carbonyl chloride(2.73 g, 10.52 mmol, 1.50 equiv.), TEA (2.12 g, 21.03 mmol, 3.00equiv.). The resulting solution was stirred for 1 h at 0° C. The residuewas purified by a silica gel column, eluted with ethyl acetate/petroleumether (1:5) to afford 3.3 g (67%) ofN-[1-[3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-[1-(benzyloxy)-2-methylpropan-2-yl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamideas light yellow oil.

N-[2-[1-(benzyloxy)-2-methylpropan-2-yl]-1-[3-(benzyloxy)-2-oxo(3,3-²H₂)propyl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide:To a 50-mL round-bottom flask, was placedN-[1-[3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-[1-(benzyloxy)-2-methylpropan-2-yl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide(2.00 g, 2.85 mmol, 1.00 equiv.), MeCN (40 mL) and IBX (4.00 g, 14.25mmol, 5.00 equiv.). The resulting solution was stirred for at 50° C. 16h. The residue was purified by a silica gel column, eluted with ethylacetate/petroleum ether (1:5) to afford 1.9 g (95%) ofN-[2-[1-(benzyloxy)-2-methylpropan-2-yl]-1-[3-(benzyloxy)-2-oxo(3,3-²H₂)propyl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamideas light yellow oil.

N-[1-[3-(benzyloxy)-2-hydroxy(2,3,3-²H₃)propyl]-2-[1-(benzyloxy)-2-methylpropan-2-yl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide:N-[2-[1-(benzyloxy)-2-methylpropan-2-yl]-1-[3-(benzyloxy)-2-oxo(3,3-²H₂)propyl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide(1.90 g, 2.72 mmol, 1.00 equiv) was dissolved in CD₃OD (30 mL). NaBD₄(114 mg, 2.72 mmol, 1.00 equiv.) was added to this solution at 0° C. Theresulting solution was stirred at room temperature for 1 h. The reactionwas quenched by D₂O (3 mL) and evaporated with the temperature below 40°C. The residue was extracted with 3×50 mL EtOAc. The organic layers werecombined, dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was purified by a silica gel column, eluted withethyl acetate/petroleum ether (1:5) to afford 1.3 g (68%) ofN-[1-[3-(benzyloxy)-2-hydroxy(2,3,3-²H₃)propyl]-2-[1-(benzyloxy)-2-methylpropan-2-yl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamideas light yellow oil.

(R)1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)-N-[1-[2,3-dihydroxy(2,3,3-²H₃)propyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl]cyclopropane-1-carboxamide

To a 50-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of H₂, was placedN-[1-[3-(benzyloxy)-2-hydroxy(2,3,3-²H₃)propyl]-2-[1-(benzyloxy)-2-methylpropan-2-yl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide(1.3 g, 1.85 mmol, 1.00 equiv.), dry Pd/C (1 g) and MeOH (10 mL, 6MHCl). The resulting mixture was stirred at room temperature for 2 huntil LCMS indicated the completion of the reaction. The solids werefiltered out and the organic phase was concentrated under vacuum. Theresidue was purified by prep-HPLC with the following conditions: Column,XBridge Prep C18 OBD Column 19×150 mm, Sum; mobile phase and Gradient,Phase A: Waters (0.1% FA), Phase B: ACN; Detector, UV 254 nm and thenfurther purified by Prep-SFC with the following conditions: Column,CHIRALPAK-IC-SFC-02, 5 cm*25 cm; mobile phase, CO₂ (50%), methanol(50%); Detector, UV 254 nm to afford 102.6 mg (10.6%) of(R)1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)-N-[1-[2,3-dihydroxy(2,3,3-²H₃)propyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl]cyclopropane-1-carboxamideas a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ: 8.33 (s, 1H), 7.53(s, 1H), 7.45-7.28 (m, 4H), 6.22 (s, 1H), 5.00 (brs, 1H), 4.88 (brs,1H), 4.78-4.75 (m, 1H), 4.42-4.39 (m, 1H), 4.14-4.08 (m, 1H), 3.64-3.59(m, 2H), 1.47-1.46 (m, 2H), 1.35-1.32 (m, 6H), 1.14-1.13 (m, 2H). LCMS:m/z=524.1 [M+H]+.

EXAMPLE 5(R)1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)-N-[1-[2,3-dihydroxy(2,3,3-²H₃)propyl]-6-fluoro-2-(1-hydroxy-2-methyl(1,1-²H₂)propan-2-yl)-1H-indol-5-yl]cyclopropane-1-carboxamide

1-N-[3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-[4-(benzyloxy)-3,3-dimethyl(1,1-²H₂)but-1-yn-1-yl]-5-fluorobenzene-1,4-diamine

To a 250-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed4-(3-(benzyloxy)-2-hydroxy(3,3-²H₂)propylamino)-5-bromo-2-fiuorobenzenaminium4-methylbenzenesulfonate (2.8 g, 5.16 mmol, 1.00 equiv.),([2,2-dimethylbut-3-yn-1-yl]oxymethyl)benzene (1.46 g, 7.75 mmol, 1.50equiv), potassium carbonate (2.13 g, 15.05 mmol, 3.00 equiv), Pd(OAc)₂(46.4 mg, 0.21 mmol, 0.04 equiv), dppb (132.2 mg, 0.31 mmol, 0.06equiv.) and MeCN (50 mL). The resulting solution was stirred for 16 h at80° C. The solids were filtered out. The organic phase was dried overanhydrous sodium sulfate and concentrated under vacuum. The residue waspurified by a silica gel column, eluted with ethyl acetate/petroleumether (1:5). To afford 0.84 g (30%) of1-N-[3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-[4-(benzyloxy)-3,3-dimethyl(1,1-²H₂)but-1-yn-1-yl]-5-fluorobenzene-1,4-diamineas light yellow oil.

1-[5-amino-2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-6-fluoro-1H-indol-1-yl]-3-(benzyloxy)(3,3-²H₂)propan-2-ol

To a 40-mL sealed tube purged and maintained with an inert atmosphere ofnitrogen, was placed1-N-[3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-[4-(benzyloxy)-3,3-dimethyl(1,1-²H₂)but-1-yn-1-yl]-fluorobenzene-1,4-diamine(840 mg, 1.75 mmol, 1.00 equiv.), MeCN (10 mL), Pd(MeCN)₂Cl₂ (68.3 mg,0.26 mmol, 0.15 equiv.). The resulting solution was stirred for 16 h at80° C. The solids were filtered and the filtrate was concentrated toafford 845 mg of the crude product, which was used without furtherpurification.

N-[1-[(2R)-3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-5-fluoro-1H-indol-6-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide

To a 50-mL round-bottom flask, was placed1-[5-amino-2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-6-fluoro-1H-indol-1-yl]-3-(benzyloxy)(3,3-²H₂)propan-2-ol(845 mg, 1.75 mmol, 1.00 equiv.), dichloromethane (15 mL),1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carbonyl chloride(683 mg, 2.63 mmol, 1.50 equiv.), TEA (530 mg, 5.26 mmol, 3.00 equiv.).The resulting solution was stirred for 1 h at 0° C. The residue waspurified by a silica gel column, eluted with ethyl acetate/petroleumether (1:5) to afford 825 mg (67%) ofN-[1-[3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamideas light yellow oil.

N-[2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-1-[3-(benzyloxy)-2-oxo(3,3-²H₂)propyl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide

To a 50-mL round-bottom flask, was placedN-[1-[3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide(825 mg, 0.71 mmol, 1.00 equiv.), MeCN (20 mL) and IBX (1.65 g, 3.55mmol, 5.00 equiv.). The resulting solution was stirred for at 50° C. 16h. The residue was purified by a silica gel column, eluted with ethylacetate/petroleum ether (1:5) to afford 784 mg (95%) ofN-[2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-1-[3-(benzyloxy)-2-oxo(3,3-²H₂)propyl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamideas light yellow oil.

N-[1-[3-(benzyloxy)-2-hydroxy(2,3,3-²H₃)propyl]-2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide

N-[2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-1-[3-(benzyloxy)-2-oxo(3,3-²H₂)propyl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide(784 mg, 1.12 mmol, 1.00 equiv.) was dissolved in CD₃OD (10 mL). NaBD₄(47 mg, 1.12 mmol, 1.00 equiv.) was added to this solution at 0° C. Theresulting solution was stirred at room temperature for 1 h. The reactionwas quenched by D₂O (3 mL) and evaporated with the temperature below 40°C. The residue was extracted with 3×10 mL EA. The organic layers werecombined, dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was purified by a silica gel column, eluted withethyl acetate/petroleum ether (1:5) to afford 530 mg (65%) ofN-[1-[3-(benzyloxy)-2-hydroxy(2,3,3-²H₃)propyl]-2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamideas light yellow oil.

(R)1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)-N-[1-[2,3-dihydroxy(2,3,3-²H₃)propyl]-6-fluoro-2-(1-hydroxy-2-methyl(1,1-²H₂)propan-2-yl)-1H-indol-5-yl]cyclopropane-1-carboxamide

To a 50-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of H₂, was placedN-[1-[3-(benzyloxy)-2-hydroxy(2,3,3-²H₃)propyl]-2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide(530 g, 0.75 mmol, 1.00 equiv), dry Pd/C (300 mg) and MeOH (5 mL, 6MHCl). The resulting mixture was stirred at room temperature for 2 huntil LCMS indicated the completion of the reaction. The solids werefiltered out and the organic phase was concentrated under vacuum. Theresidue was purified by prep-HPLC with the following conditions: Column,XBridge Prep C18 OBD Column 19×150 mm, 5 um; mobile phase and Gradient,Phase A: Waters (0.1% FA), Phase B: ACN; Detector, UV 254 nm and thenfurther purified by Prep-SFC with the following conditions: Column,CHIRALPAK-IC-SFC-02, 5 cm*25 cm; mobile phase, CO₂ (50%), methanol(50%); Detector, UV 254 nm to afford 23.8 mg (6.1%) of(R)1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)-N-[1-[2,3-dihydroxy(2,3,3-²H₃)propyl]-6-fluoro-2-(1-hydroxy-2-methyl(1,1-²H₂)propan-2-yl)-1H-indol-5-yl]cyclopropane-1-carboxamideas a light yellow solid. ¹H NMR (400 MHz, Methanol-d₄) δ: 7.63 (d, J=7.2Hz, 1H), 7.44-7.32 (m, 2H), 7.27-7.24 (m, 2H), 6.32 (s, 1H), 4.43-4.29(m, 2H), 1.66-1.64 (m, 2H), 1.47 (s, 3H), 1.38 (s, 3H), 1.22-1.21 (m,2H). LCMS: m/z=526.2 [M+H]⁺.

EXAMPLE 6(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methyl(1,1-²H₂)propan-2-yl)-1H-indol-5-yl)(²H₄)cyclopropanecarboxamide

1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H₄)cyclopropane-1-carbonitrile

To a 25-mL round-bottom flask, was placed potassium hydroxide (4.26 g,75.92 mmol, 4.99 equiv.), D₂O (4.26 g),2-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)acetonitrile (3 g, 15.22 mmol,1.00 equiv.), 1-bromo-2-chloro(²H₄)ethane (4.34 g, 29.44 mmol, 1.50equiv.), Bu₄NBr (98 g, 305.30 mmol, 0.02 equiv.). The resulting solutionwas stirred for 48 h at 70° C. The resulting solution was extracted with3×50 mL of ethyl acetate and the organic layers were combined, driedover anhydrous sodium sulfate and concentrated under vacuum to afford 3g (87%) of1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H₄)cyclopropane-1-carbonitrileas red oil.

1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H)cyclopropane-1-carboxylic

To a 100-mL round-bottom flask, was placed 6 M sodium hydroxide (18 mL),ethanol (15 mL),1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H₄)cyclopropane-1-carbonitrile(3 g, 13.20 mmol, 1.00 equiv.). The resulting solution was stirred for16 h at 80° C. The resulting mixture was concentrated under vacuum. ThepH value of the solution was adjusted to 3-4 with hydrogen chloride (3mol/L). The reaction mixture was cooled. The solids were collected byfiltration to afford 2.5 g (77%) of1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H₄)cyclopropane-1-carboxylicacid as a light yellow solid.

1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H₄)cyclopropane-1-carbonylchloride

To a 50-mL round-bottom flask, was placed1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H₄)cyclopropane-1-carboxylicacid (2.5 g, 10.15 mmol, 1.00 equiv), thionyl chloride (6 g, 50.85 mmol,5.00 equiv.), toluene (10 mL). The resulting solution was stirred for 3h at 65° C. The resulting mixture was concentrated under vacuum toafford 2.68 g (99%) of1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H₄)cyclopropane-1-carbonylchloride as a light yellow solid.

N-[1-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-5-fluoro-2-[1-hydroxy-2-methyl(1,1-²H₂)propan-2-yl]-1H-indol-6-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H₄)cyclopropane-1-carboxamide

To a 50-mL round-bottom flask, was placed(2R)-1-[5-amino-2-[1-(benzyloxy)-2-methyl(1,1-²H₂)propan-2-yl]-6-fluoro-1H-indol-1-yl]-3-(benzyloxy)propan-2-ol(900 mg, 1.88 mmol, 1.00 equiv.), TEA (400 mg, 3.95 mmol, 2.00 equiv.),dichloromethane (20 mL),1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H₄)cyclopropane-1-carbonylchloride (745 mg, 2.82 mmol, 1.50 equiv.). The resulting solution wasstirred for 1 h at 0° C. The residue was purified by a silica gelcolumn, eluted with ethyl acetate/petroleum ether (1:5) to afford 500 mg(43%) ofN-[1-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-5-fluoro-2-[1-hydroxy-2-methyl(1,1-²H₂)propan-2-yl]-1H-indol-6-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H₄)cyclopropane-1-carboxamideas light yellow oil.

(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methyl(1,1-²H₂)propan-2-yl)-1H-indol-5-yl)(²H₄)cyclopropanecarboxamide

To a 100-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of H₂, was placedN-[1-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-5-fluoro-2-[1-hydroxy-2-methyl(1,1-²H₂)propan-2-yl]-1H-indol-6-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H₄)cyclopropane-1-carboxamide(400 mg, 0.77 mmol, 1.00 equiv.) dry Pd/C (300 mg) and MeOH (5 mL, 6MHCl). The resulting mixture was stirred at room temperature for 2 huntil LCMS indicated the completion of the reaction. The solids werefiltered out and the resulting mixture was concentrated under vacuum.The residue was purified by prep-HPLC with the following conditions:Column, XBridge Prep C18 OBD Column 19×150 mm, Sum; mobile phase andGradient, Phase A: Waters (0.05% TFA), Phase B: ACN; Detector, UV 254 nmto afford 119.2 mg (32%) of(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methyl(1,1-²H₂)propan-2-yl)-1H-indol-5-yl)(²H₄)cyclopropanecarboxamide as a light yellow solid. ¹H NMR (400 MHz,DMSO-d₆) δ: 8.32 (s, 1H), 7.54 (s, 1H), 7.43-7.38 (m, 2H), 7.34-7.31 (m,2H), 6.22 (s, 1H), 4.42-4.39 (m, 1H), 4.14-4.08 (m, 1H), 3.91 (brs, 1H),3.47-3.38 (m, 2H), 1.36 (s, 3H), 1.32 (s, 3H). LCMS: m/z=527.3[M+H]⁺.

EXAMPLE 7 [3-chloro-3-(²H₆)methyl(1,1-H)but-1-yn-1-yl]trimethylsilane

N-[1-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-5-fluoro-2-[1-hydroxy-2-methyl(²H₆)propan-2-yl]-1H-indol-6-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H₄)cyclopropane-1-carboxamide

To a 50-mL round-bottom flask, was placed(2R)-1-[5-amino-2-[1-(benzyloxy)-2-bismethyl(²H₆)propan-2-yl]-6-fluoro-1H-indol-1-yl]-3-(benzyloxy)propan-2-ol(900 mg, 1.88 mmol, 1.00 equiv), TEA (400 mg, 3.95 mmol, 2.00 equiv.),dichloromethane (20 mL),1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H₄)cyclopropane-1-carbonylchloride (745 mg, 2.82 mmol, 1.50 equiv.). The resulting solution wasstirred for 1 h at 0° C. The residue was purified by a silica gelcolumn, eluted with ethyl acetate/petroleum ether (1:5) to afford 500 mg(39%) ofN-[1-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-5-fluoro-2-[1-hydroxy-2-methyl(²H₆)propan-2-yl]-1H-indol-6-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H₄)cyclopropane-1-carboxamideas light yellow oil.

[3-chloro-3-(²H₆)methyl(1,1-H)but-1-yn-1-yl]trimethylsilane

To a 100-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of H₂, was placedN-[1-[(2R)-3-(benzyloxy)-2-hydroxypropyl]-5-fluoro-2-[1-hydroxy-2-methyl(²H₆)propan-2-yl]-1H-indol-6-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(²H₄)cyclopropane-1-carboxamide(500 mg, 0.68 mmol, 1.00 equiv.) dry Pd/C (500 mg) and MeOH (10 mL, 6 MHCl). The resulting mixture was stirred at room temperature for 2 huntil LCMS indicated the completion of the reaction. The solids werefiltered out and the resulting mixture was concentrated under vacuum.The residue was purified by prep-HPLC with the following conditions:Column, XBridge Prep C18 OBD Column 19×150 mm, Sum; mobile phase andGradient, Phase A: Waters (0.1% FA), Phase B: ACN; Detector, UV 254 nmto afford 199.2 mg (55%) of(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methyl(²H₆)propan-2-yl)-1H-indol-5-yl)(²H₄)cyclopropanecarboxamide as a light yellow solid. ¹H NMR (400 MHz,DMSO-d6) δ: 8.31 (s, 1H), 7.53 (s, 1H), 7.43-7.38 (m, 2H), 7.34-7.31 (m,2H), 6.21 (s, 1H), 5.02-5.01 (m, 1H), 4.92-4.90 (m, 1H), 4.76-4.73 (m,1H), 4.44-4.39 (m, 1H), 4.13-4.07 (m, 1H), 3.90 (brs, 1H), 3.64-3.54 (m,2H), 3.47-3.31 (m, 2H). LCMS: m/z=531.2[M+H]+.

EXAMPLE 8(R)1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)-N-[1-[2,3-dihydroxy(3,3-²H₂)propyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl]cyclopropane-1-carboxamide

(R)1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)-N-[1-[2,3-dihydroxy(3,3-²H₂)propyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl]cyclopropane-1-carboxamide

To a 50-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of H₂, was placedN-[1-[3-(benzyloxy)-2-hydroxy(3,3-²H₂)propyl]-2-[1-(benzyloxy)-2-methylpropan-2-yl]-6-fluoro-1H-indol-5-yl]-1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)cyclopropane-1-carboxamide(1.3 g, 1.85 mmol, 1.00 equiv), dry Pd/C (1 g) and MeOH (10 mL, 6 MHCl). The resulting mixture was stirred at room temperature for 2 huntil LCMS indicated the completion of the reaction. The solids werefiltered out and the organic phase was concentrated under vacuum. Theresidue was purified by prep-HPLC with the following conditions: Column,XBridge Prep C18 OBD Column 19×150 mm, Sum; mobile phase and Gradient,Phase A: Waters (0.1% FA), Phase B: ACN; Detector, UV 254 nm and thenfurther purified by Prep-SFC with the following conditions: Column,CHIRALPAK-IC-SFC-02, 5 cm*25 cm; mobile phase, CO₂ (50%), methanol(50%); Detector, UV 254 nm to afford 111.8 mg (11.5%) of(R)1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)-N-[1-[2,3-dihydroxy(3,3-²H₂)propyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl]cyclopropane-1-carboxamideas a light yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.33 (s, 1H), 7.53(s, 1H), 7.45-7.28 (m, 4H), 6.22 (s, 1H), 5.05-4.97 (m, 1H), 4.89-4.87(m, 1H), 4.78-4.76 (m, 1H), 4.42-4.38 (m, 1H), 4.16-4.05 (m, 1H),3.93-3.85 (m, 1H), 3.64-3.57 (m, 2H), 1.48-1.46 (m, 2H), 1.36-1.33 (m,6H), 1.14-1.12 (m, 2H). LCMS: m/z=523.2 [M+H]⁺.

The following compounds can generally be made using the methodsdescribed above. It is expected that these compounds when made will haveactivity similar to those described in the examples above.

Changes in the metabolic properties of the compounds disclosed herein ascompared to their non-isotopically enriched analogs can be shown usingthe following assays. Compounds listed above which have not yet beenmade and/or tested are predicted to have changed metabolic properties asshown by one or more of these assays as well.

Biological Activity Assays

In Vitro Liver Microsomal Stability Assay

Human liver microsomal stability assays were conducted at 2 mg per mLliver microsome protein with an NADPH-generating system consisting ofNADP (1 mM, pH 7.4), glucose 6-phosphate (5 mM, pH 7.4) and glucose6-phosphate dehydrogenase (I unit/mL). Test compounds were prepared assolutions in DMSO and added to the assay mixture (1 uM, finalconcentration in incubation) and incubated at 37±1° C. Reactions wereinitiated with addition of the test compounds and stopped at 0, 60, 120or 240 min after test article addition with stop reagent, acetonitrile.Samples were centrifuged (920×g for 10 min at 10° C.) in 96-well plates.Supernatant fractions were analyzed by LC-MS/MS to determine the percentremaining and estimate the degradation half-life of the test compounds.Results are given below.

Clearance % half-life % Example change over d0 change over d0 Example 10.0 0.0 Example 2 −66.7 200.0 Example 3 −11.9 13.3 Example 4 −4.8 4.8Example 5 −71.4 250.3 Example 6 −73.8 281.8 Example 7 4.8 −4.2 Example 8−4.8 4.8

Liver microsomal stability assays may also be conducted at 1 mg per mLliver microsome protein with an NADPH-generating system in 2% NaHCO₃(2.2 mM NADPH, 25.6 mM glucose 6-phosphate, 6 units per mL glucose6-phosphate dehydrogenase and 3.3 mM MgCl₂). Test compounds are preparedas solutions in 20% acetonitrile-water and added to the assay mixture(final assay concentration 5 microgram per mL) and incubated at 37° C.Final concentration of acetonitrile in the assay should be <1%. Aliquots(504) are taken out at times 0, 15, 30, 45, and 60 min, and diluted withice cold acetonitrile (200 μL) to stop the reactions. Samples arecentrifuged at 12,000 RPM for 10 min to precipitate proteins.Supernatants are transferred to microcentrifuge tubes and stored forLC/MS/MS analysis of the degradation half-life of the test compounds.

In Vitro Metabolism Using Human Cytochrome P450 Enzymes

The cytochrome P450 enzymes are expressed from the corresponding humancDNA using a baculovirus expression system (BD Biosciences, San Jose,Calif.). A 0.25 milliliter reaction mixture containing 0.8 milligramsper milliliter protein, 1.3 millimolar NADP⁺, 3.3 millimolarglucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase, 3.3millimolar magnesium chloride and 0.2 millimolar of a compound ofFormula I, the corresponding non-isotopically enriched compound orstandard or control in 100 millimolar potassium phosphate (pH 7.4) isincubated at 37° C. for 20 min. After incubation, the reaction isstopped by the addition of an appropriate solvent (e.g., acetonitrile,20% trichloroacetic acid, 94% acetonitrile/6% glacial acetic acid, 70%perchloric acid, 94% acetonitrile/6% glacial acetic acid) andcentrifuged (10,000 g) for 3 min. The supernatant is analyzed byHPLC/MS/MS. Compounds disclosed herein are expected to have activity inthis assay as demonstrated by reduced metabolism by one or morecytochrome P450 enzymes of deuterated compound as compared to thenon-isotopically enriched compound.

Cytochrome P₄₅₀ Standard CYP1A2 Phenacetin CYP2A6 Coumarin CYP2B6[¹³C]-(S)-mephenytoin CYP2C8 Paclitaxel CYP2C9 Diclofenac CYP2C19[¹³C]-(S)-mephenytoin CYP2D6 (+/−)-Bufuralol CYP2E1 Chlorzoxazone CYP3A4Testosterone CYP4A [¹³C]-Lauric acidIn Vitro Hepatocyte Stability Assay

Test compounds are typically prepared as solutions with a minimum oforganic solvent, added to the assay mixture (10 uM, final concentrationin incubation with no more than 0.1% acetonitrile, methanol, and/or 0.2%dimethyl sulfoxide) and incubated at 37° C. in 5% CO₂. Reactions areinitiated with addition of the test compounds to cryopreserved human orrat cryopreserved hepatocyte culture suspensions for 0, 0.5, 1, 2, 3,and 4 hours in 48-well plates. At each time point, incubation mixturesare extracted with 3 volumes of ice-cold acetonitrile: ethanol (3:1,v:v) containing appropriate internal standards. Extracts are transferredto 96-well plates at stored at −20° C. Extracts are subsequentlyanalyzed by LC-MS/MS to determine the percent remaining and estimate thedegradation half-life of the test compounds. In addition, metabolitesare identified with LC/MS using a high resolution full scan method.Relative abundance of metabolites of interested are determined via UVspectra or full scan LC-MS with accurate mass or with LC-MRM. Therelative abundance of metabolite to the total of all metabolites plusunchanged drug is determined. Compounds disclosed herein are expected tohave activity in this assay as demonstrated by reduced clearance andincreased degradation half-life of deuterated compound as compared tothe non-isotopically enriched compound.

Monoamine Oxidase A Inhibition and Oxidative Turnover

The procedure is carried out using the methods described by Weyler,Journal of Biological Chemistry 1985, 260, 13199-13207, which is herebyincorporated by reference in its entirety. Monoamine oxidase A activityis measured spectrophotometrically by monitoring the increase inabsorbance at 314 nm on oxidation of kynuramine with formation of4-hydroxyquinoline. The measurements are carried out, at 30° C., in 50mM NaP_(i) buffer, pH 7.2, containing 0.2% Triton X-100 (monoamineoxidase assay buffer), plus 1 mM kynuramine, and the desired amount ofenzyme in 1 mL total volume.

Monoamine Oxidase B Inhibition and Oxidative Turnover

The procedure is carried out as described in Uebelhack,Pharmacopsychiatry 1998, 31(5), 187-192, which is hereby incorporated byreference in its entirety.

Assays for Detecting and Measuring ΔF508-CFTR Correction Properties ofCompounds

The procedure is carried out as described in WO 2014014841, which ishereby incorporated by reference in its entirety. Sequence analysis ofthe CFTR gene of CF chromosomes has revealed a variety ofdisease-causing mutations. The most prevalent mutation is a deletion ofphenylalanine at position 508 of the CFTR amino acid sequence, and iscommonly referred to as AF508-CFTR. This mutation occurs inapproximately 70 percent of the cases of cystic fibrosis and isassociated with a severe disease. The deletion of residue 508 inAF508-CFTR prevents the nascent protein from folding correctly. Thisresults in the inability of the mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, the number of channelspresent in the membrane is far less than observed in cells expressingwild-type CFTR. In addition to impaired trafficking, the mutationresults in defective channel gating. Together, the reduced number ofchannels in the membrane and the defective gating lead to reduced aniontransport across epithelia leading to defective ion and fluid transport.

Optical membrane potential assays may utilizes voltage-sensitive FRETsensors. These voltage sensitive assays are based on the change influorescence resonant energy transfer (FRET) between themembrane-soluble, voltage-sensitive dye, DiSBAC₂(3), and a fluorescentphospholipid, CC2-DMPE, which is attached to the outer leaflet of theplasma membrane and acts as a FRET donor. Changes in membrane potential(Vm) cause the negatively charged DiSBAC₂(3) to redistribute across theplasma membrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission were monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

Solutions. Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂1, HEPES 10, pH 7.4 with NaOH. Chloride-free bath solution: Chloridesalts in Bath Solution #1 are substituted with gluconate salts.CC2-DMPE: Prepared as a 10 mM stock solution in DMSO and stored at −20°C. DiSBAC₂(3): Prepared as a 10 mM stock in DMSO and stored at −20° C.

Cell Culture. NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR areused for optical measurements of membrane potential. The cells aremaintained at 37° C. in 5% CO₂ and 90% humidity in Dulbecco's modifiedEagle's medium supplemented with 2 mM glutamine, 10% fetal bovine serum,1×NEAA, β-ME, 1×pen/strep, and 25 mM HEPES in 175 cm2 culture flasks.For all optical assays, the cells were seeded at 30,000/well in 384-wellmatrigel-coated plates and cultured for 2 hrs at 37° C. before culturingat 27° C. for 24 hrs for the potentiator assay. For the correctionassays, the cells are cultured at 27° C. or 37° C. with and withoutcompounds for 16-24 hours.

To identify small molecules that correct the trafficking defectassociated with ΔF508-CFTR; a single-addition HTS assay format may beused. The cells are incubated in serum-free medium for 16 hrs at 37° C.in the presence or absence (negative control) of test compound. As apositive control, cells plated in 384-well plates are incubated for 1hrs at 27° C. to “temperature-correct” ΔF508-CFTR. The cells aresubsequently rinsed 3× with Krebs Ringers solution and loaded with thevoltage-sensitive dyes. To activate ΔF508-CFTR, 10 μM forskolin and theCFTR potentiator, genistein (20 μM), are added along with Cl′-freemedium to each well. The addition of Cl⁻free medium promoted CI″ effluxin response to AF508-CFTR activation and the resulting membranedepolarization is optically monitored using the FRET-basedvoltage-sensor dyes.

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat may be used. During the first addition, a Cl⁻free medium with orwithout test compound is added to each well. After 22 sec, a secondaddition of Cl⁻-free medium containing 2⁻¹⁰ μM forskolin is added toactivate ΔF508-CFTR. The extracellular CI″ concentration following bothadditions is 28 mM, which promoted Cl⁻ efflux in response to ΔF508-CFTRactivation and the resulting membrane depolarization is opticallymonitored using the FRET-based voltage-sensor dyes.

Compounds disclosed herein are expected to have activity in this assay.

Animal Models of Cystic Fibrosis

Although CFTR correction is typically monitored by the restoration of acAMP-mediated Cl⁻ current, an animal model could be used to establishthe clinically relevant outcome measurement, such as the amount of CFTRcorrection that is necessary to prevent inflammation and infection.

CFTR-deficient (Rogers, C. S. et al. J. Clin. Invest. 118, 1571-1577,2008) and ΔF508/ΔF508 pigs (Ostedgaard, L S. et al, Sci. Transl. Med. 74ra24, 2011) spontaneously develop lung disease which is characterized byinflammation, mucus overproduction, airway obstruction and infection.These CFTR-deficient pigs manifest the predicted defect in chloride andbicarbonate transport that is typical of human CFTR mutations, but theydo not hyperabsorb sodium nor do they show diminished amounts of airwaysurface fluid. These results suggest that decreased hydration of airwaysurface fluid may not be central to the development of infection andinflammation (Chen, J. H. et al. Cell 143, 911-923, 2010). (Similarly,the cystic fibrosis ferret also develops lung infection very early inlife, which is severe enough to require antibiotic treatment (Sun, X. etal. J. Clin. Invest 120, 3149-3160, 2010).) Such models support thehypothesis that there is a direct role of CFTR in mucosal immunitybeyond its contribution to the hydration of the airway surface fluids.These animal models provide useful model systems to test thepharmacologic agents which are described herein. Compounds disclosedherein are expected to have activity in this assay; measures of efficacyinclude decreased inflammation, mucus overproduction, airway obstructionand infection.

Clinical Trials in Cystic Fibrosis

Methods for pre-clinical testing of pharmacologic agents with potentialactivity in cystic fibrosis patients are described in Döring et al. J.Cystic Fibrosis 6, 85-99, 2007. Study design options for phase II andphase III studies involving cystic fibrosis patients are provided,including required patient numbers, safety issues and surrogate endpoint parameters for drugs, tested for different disease manifestations.Compounds disclosed herein are expected to effectively treat cysticfibrosis and/or its symptoms.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A compound of Formula I:

or a salt thereof, wherein: R₁-R₃ and R₆-R₂₃ are, independently,hydrogen or deuterium; R₄-R₅ are, independently, —CH₃, —CH₂D, —CD₂H, or—CD₃; at least one of R₁-R₂₃ is deuterium or contains deuterium; and atleast one of R₁-R₂₃ independently has deuterium enrichment of no lessthan about 10%.
 2. The compound, or a salt thereof, of claim 1, whereinR₁, R₆, R₉, and R₁₆ are hydrogen.
 3. The compound, or a salt thereof, ofclaim 2, wherein R₂ and R₃ are deuterium.
 4. The compound, or a saltthereof, of claim 2, wherein R₄ and R₅ are —CD₃.
 5. The compound, or asalt thereof, of claim 2, wherein R₇ and R₈ are deuterium.
 6. Thecompound, or a salt thereof, of claim 2, wherein R₇, R₈, and R₁₀ aredeuterium.
 7. The compound, or a salt thereof, of claim 2, wherein R₂,R₃, R₇, R₈, and R₁₀ are deuterium.
 8. The compound, or a salt thereof,of claim 2, wherein R₁₇-R₂₀ are deuterium.
 9. The compound, or a saltthereof, of claim 2, wherein R₂, R₃, and R₁₇-R₂₀ are deuterium.
 10. Thecompound, or a salt thereof, of claim 2, wherein R₇, R₈, and R₁₇-R₂₀ aredeuterium.
 11. The compound, or a salt thereof, of claim 2, wherein R₇,R₈, R₁₀, and R₁₇-R₂₀ are deuterium.
 12. The compound, or a salt thereof,of claim 2, wherein R₂, R₃, R₇, R₈, and R₁₇-R₂₀ are deuterium.
 13. Thecompound, or a salt thereof, of claim 2, wherein R₂, R₃, R₇, R₈, R₁₀,and R₁₇-R₂₀ are deuterium.
 14. The compound, or a salt thereof, of claim2, wherein R₂₁-R₂₃ and R₁₃-R₁₅ are hydrogen.
 15. The compound, or a saltthereof, of claim 1 wherein at least one of R₁-R₂₃ independently hasdeuterium enrichment selected from of no less than about 50%, of no lessthan about 90%, or of no less than about 98%.
 16. The compound, or asalt thereof, as recited in claim 1, wherein the compound is:

or a salt thereof.
 17. A pharmaceutical composition comprising thecompound, or a salt thereof, of claim 1 and a pharmaceuticallyacceptable carrier.
 18. A method of treating a cystic fibrosistransmembrane conductance regulator-mediated disorder, comprisingadministering a therapeutically effective amount of the compound, or asalt thereof, of claim 1 to a patient in need thereof.
 19. The method ofclaim 18, wherein the disorder is cystic fibrosis, sarcoglycanopathies,Brody's disease, cathecolaminergic polymorphic ventricular tachycardia,limb girdle muscular dystrophy, asthma, smoke induced chronicobstructive pulmonary disorder, chronic bronchitis, rhinosinusitis,constipation, pancreatitis, pancreatic insufficiency, male infertilitycaused by congenital bilateral absence of the vas deferens, mildpulmonary disease, idiopathic pancreatitis, allergic bronchopulmonaryaspergillosis, liver disease, hereditary emphysema, hereditaryhemochromatosis, coagulation-fibrinolysis deficiencies, such as proteinC deficiency, type 1 hereditary angioedema, lipid processingdeficiencies, such as familial hypercholesterolemia, type 1chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, suchas I-cell disease/pseudo-Hurler, mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,polyendocrinopathy/hyperinsulinemia, diabetes mellitus, Laron dwarfism,myeloperoxidase deficiency, primary hypoparathyroidism, melanoma,glycanosis CDG type 1, congenital hyperthyroidism, osteogenesisimperfecta, hereditary hypofibrinogenemia, ACT deficiency, diabetesinsipidus (DI), neurohypophyseal DI, nephrogenic DI, Charcot-Marie toothsyndrome, Pelizaeus-Merzbacher disease, neurodegenerative diseases suchas Alzheimer's disease, Parkinson's disease, amyotrophic lateralsclerosis, progressive supranuclear palsy, Pick's disease, polyglutamineneurological disorders such as Huntington's, spinocerebellar ataxia typeI, spinal and bulbar muscular atrophy, dentatombral pallidoluysian, andmyotonic dystrophy, as well as spongifiorm encephalopathies, such ashereditary Creutzfeldt-Jakob disease, Fabry disease,Gerstrnarm-Straussler-Scheinker syndrome, chronic obstructive pulmonarydisorder, dry-eye disease, or Sjogren's disease, osteoporosis,osteopenia, bone healing and bone growth, Gorham's Syndrome, chloridechannelopathies such as myotonia congenita, Bartter's syndrome type III,Dent's disease, hyperekplexia, epilepsy, lysosomal storage disease,Angelman syndrome, and primary ciliary dyskinesia (PCD), a term forinherited disorders of the structure and/or function of cilia, includingPCD with situs inversus, PCD without situs inversus, and ciliaryaplasia.
 20. The method of claim 19, further comprising administering anadditional therapeutic agent.